bluuebunny/crossref_metadata_2025 huggingface dataset:
3‐O‐sulfation provides high affinity binding to neuropilin‐1 and modulates endothelial cell sprouting and neuronal growth cone collapse (607.7)
Authors: Thacker Bryan , Lawrence Roger , Seamen Emylie , Liu JIan , Esko Jeffrey
Published: June 2021

Heparan sulfate structural heterogeneity is driven, in part, by the placement of sulfate groups at various positions in the polysaccharide. While many ligands bind to heparan sulfate without strict requirements for the positions of sulfate groups, binding of a small number of known ligands is influenced by the presence of a sulfate at the C3 position of a glucosamine residue. In mammals, seven enzymes can catalyze the addition of 3‐O‐sulfate groups, suggesting the possibility of other, previously unidentified, ligands whose binding is influenced by 3‐O‐sulfated sequences. To identify these ligands, affinity matrices were created using CHO‐S heparan sulfate, with and without modification by recombinant 3‐O‐sulfotransferases. Serum was fractionated on the matrices and several proteins binding specifically to the 3‐O‐sulfated resins were identified by mass spectrometry. Neuropilin‐1, a modulator of angiogenesis and axonal guidance, bound specifically to affinity matrices with 3‐O‐sulfation. In addition, high affinity binding of neuropilin‐1 to the cell surface required 3‐O‐sulfation. In cell culture, 3‐O‐sulfation modulated two neuropilin‐1 dependent processes. (1) In VEGF‐induced endothelial cell sprouting, 3‐O‐sulfation endowed CHO‐S heparan sulfate with greater inhibitory potential than native heparan sulfate. (2) In semaphorin‐3a‐induced neuronal growth cone collapse, a 3‐O‐sulfated heparan sulfate dodecamer was a potent inhibitor of collapse. An identical dodecamer lacking 3‐O‐sulfation inhibited with much less efficiency. Furthermore, genetic removal of a 3‐O‐sulfotransferase (Hs3st‐2) attenuated the response of the growth cone to semaphorin‐3a. In conclusion, these results demonstrate that 3‐O‐sulfation endows heparan sulfate with enhanced affinity to neuropilin‐1 and modulates two neuropilin‐1 dependent processes, angiogenesis and axonal guidance.

Grant Funding Source: Supported by GM93131 and T32CA067754
Stress‐activated signaling in endothelial cells (609.21)
Authors: Rowan Aislinn , Farwell Sara , Lowe‐Krentz Linda
Published: June 2021

Endothelial cells (ECs) form the interior layer of blood vessels, creating a selectively permeable barrier. Damage to this monolayer triggers inflammatory responses which may eventually lead to vascular disease, making it relevant to characterize EC reactions to inflammatory stress. Pro‐inflammatory cytokine tumor necrosis factor‐α (TNF‐α) activates several pathways in target cells, which interact to determine cell fate. Binding of TNF‐α to its receptor causes activation of certain caspases, advancing apoptosis. c‐jun N‐terminal kinase (JNK) is activated as well, regulating transcription factor activity and possibly promoting an apoptotic response. Also activated is nuclear factor κ‐light‐chain‐enhancer of activated B‐cells (NFκB), a transcription factor which has pro‐survival effects, partially due to interference with both JNK and caspases. ECs encounter TNF‐α as it travels in the blood, however, while most cells are rarely exposed. Thus, we explored whether EC response to TNF‐α is similar to that of other cell types, using immunofluorescence microscopy and Western blotting to analyze TNF‐α‐induced JNK, NFκB, and caspase‐3 activity in primary bovine aortic ECs (BAOECs). NFκB is usually kept in the cytoplasm by an inhibitor; TNF‐α‐binding leads to inhibitor degradation and allows NFκB to move to the nucleus. Upon TNF‐α treatment (10 ng/mL), this translocation begins by 30 minutes, maximizes from 60 to 120 minutes, and declines thereafter. JNK activation is transient, with p‐JNK levels peaking after 30 minutes before decreasing. Excitation of BAOECs with high TNF‐α levels (50 ng/mL for 20 hours) induces caspase‐3 activation, suggesting significant apoptosis. This reaction profile resembles that of other cell types, though it is possible that there are important differences in pathway cross‐talk and communication.

Grant Funding Source: supported in part by NIH grant HL54269
Exploitation of latent allostery enables the evolution of new modes of MAP kinase regulation (609.5)
Authors: Coyle Scott , Lim Wendell
Published: June 2021

Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets coevolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5‐VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5‐FBD) that tunes mating behavior is, in contrast, not conserved in most lineages. Surprisingly, both Ste5 activator motifs could regulate MAP kinases that diverged from Fus3 prior to the emergence of Ste5, suggesting that Ste5 activators arose by exploiting latent regulatory features already present in the MAPK ancestor. The magnitude of this latent allosteric potential drifts widely among pre‐Ste5 MAP kinases, providing a pool of hidden phenotypic diversity that, when revealed by new activators, could lead to functional divergence and to the evolution of distinct signaling behaviors.
Molecular dynamics analysis of CYP2D6 polymorphisms (615.4)
Authors: DeWaal Parker , Furge Laura
Published: June 2021

The objective of the current study was to use Molecular Dynamics (MD) computational approaches to understand how the interaction of four different CYP2D6 allelic variants with common substrates (bufuralol and N‐desmethyltamoxifen) and with an inactivator (SCH66712) compare to those of reference CYP2D6 (*1). Comparative models of each allelic variant examined (*17‐2, *17‐3, *34, *53) were constructed using AMBER force fields and the starting PDB structure 3MQ4. Analysis of RMSF revealed that allelic variants displayed areas of both greater flexibility and greater rigidity compared to *1. Greater flexibility was seen in the region of helices B’ and C and helices F and G while a region of greater rigidity was clear near helix F’/G’. Samples of final structures for each allelic variant were used for docking studies using AutoDock Vina and channel analysis using CAVER.

Grant Funding Source: Supported by NIH 1R15‐GM086767‐02
A mechanism for sequential replication‐coupled destruction of CRL4‐Cdt2 substrates (616.1)
Authors: Coleman Kate , Varma Dileep , Brantley Kristen , Shibata Etsuko , Dutta Anindya , Cook Jean
Published: June 2021

The CRL4Cdt2 ubiquitin E3 ligase targets a cohort of key cell cycle regulators during both S phase and DNA repair synthesis. These substrates include the p21 CDK inhibitor, the Cdt1 origin licensing protein, and the Set8 lysine methyltransferase. The intracellular concentrations of each of these proteins has profound consequences for cell proliferation and genome stability. CRL4‐Cdt2 only targets substrates if they are bound to DNA‐loaded PCNA, coupling substrate destruction to DNA synthesis. PCNA loading does not trigger simultaneous destruction of all CRL4‐Cdt2 substrates however; Cdt1 degradation begins nearly instantaneously with PCNA loading whereas p21 degradation is significantly delayed for example. As a consequence, the substrates of CRL4‐Cdt2 are destroyed in a stereotypical order, and we have observed this order in multiple cell lines during both DNA repair synthesis and in early S phase. To identify the molecular determinants of delayed vs. immediate destruction, we generated a series of fusion proteins between Cdt1 and p21. We demonstrate that fusing just the Cdt1 PCNA‐interacting motif, a.k.a. “PIP degron,” to the p21 N‐terminus is sufficient to confer rapid rather than delayed destruction. It is not the N‐terminal location, the presence of a second PIP box, or E2 utilization, but rather the degron sequence itself that defines the order of destruction. Surprisingly, rapid vs. delayed destruction is not due to degron features previously shown to be critical for either PCNA or CRL4‐Cdt2 interaction. We generated cell lines that destroy p21 according to the Cdt1 pattern (rapid) rather than the normal p21 pattern (delayed), and we will show the effects of this conversion on DNA replication parameters during S phase and on DNA damage checkpoint responses. This study sheds light on how the substrates of a single ubiquitin E3 ligase are targeted sequentially rather than simultaneously.

Grant Funding Source: Supported by NIH GM083024 to J.G.C. and NIH F31 CA165891 to K.C.
Spent engine oil pollution and its effects on selected antioxidants of Telfairia occidentalis leaves (617.9)
Authors: Onyegeme‐Okerenta Blessing , Alozie Chukwuemeka
Published: June 2021

Spent Engine Oil (SEO) pollution and its effects on selected antioxidants [Protein oxidation (protein thiol (PSH), glutathione (GSH), and total thiol (TSH)] of Telfaria occidentalis leaves was studied. Soil sample was collected from the farmland in Ohii, Owerri West Local Government Ares of Imo State, Nigeria, weighed and polluted with different volumes of spent engine oil (SEO) to give the various percentage pollutions. The polluted soil samples were poured in several nursery bags. Three viable seeds of Telfaria occidentalis were planted in each. An unpolluted soil sample served as the control for the study. Harvest of the leaves was done after 50 days of planting and taken to the laboratory for the assessment of protein oxidation parameters (protein thiol, glutathione and total thiol) concentrations in each of the sample. Results obtained from protein thiol concentration show that there was a significant difference (P>0.05) between the control sample and the 6% polluted sample. There was a progressive reduction in the protein thiol concentration as the volume of the SEO increased. That obtained for GSH also showed a similar trend. There was no significant difference (P>0.05) between control sample and 1% sample pollution but a significant difference (P>0.05) was observed between 0% and 6%. There was also a progressive reduction in the GSH concentration as the volume of the pollutant increased. The same trend was observed for total thiol (TSH) concentration as the SEO volume increased. The mean values obtained for all the parameters (PSH, GSH and TSH) were found not to be significantly different (p>0.05) These results show that increased pollution of soil by spent engine oil have an effect on the protein oxidation parameters of edible fluted pumpkin (T. occidentalis) leaves in areas where there is indiscriminate dumping of spent oil.

Grant Funding Source: NIL
Role of undergraduate biochemistry education in protein function assignment (618.26)
Authors: Craig Paul , Dodge Greg , Bernstein Herbert
Published: June 2021

There are over 3000 unannotated structures in the PDB. Students in our labs have proposed functions for >90 of these structures using template‐based alignments with the ProMOL plugin for PyMOL, BLAST, Dali and Pfam. Subsequently the students confirmed the function of four of these structures in work that they completed with minimal supervision. First they reviewed the putative enzyme function and searched the literature for an assay for that function. If the reagents were available and relatively inexpensive, they obtained the plasmid containing the gene encoding that protein, which they expressed and purified, using metal ion chromatography of the his‐tagged protein. The purified enzyme was then characterized in vitro. The process typically requires 10 ‐ 20 weeks for an individual undergraduate student working full time. We believe that this approach can be easily implemented in undergraduate biochemistry teaching labs, where it would benefit the students and the community. The students will learn about protein expression, purification and characterization using both traditional methods (electrophoresis, UV‐Visible spectroscopy) and modern approaches (plasmid expression, single step chromatographic methods). At the same time, a small army of undergraduates could make a significant dent in the number of unannotated structures in the PDB. Supported by NIGMS 2R15GM078077‐02 and 3R15GM078077‐02S1.

Grant Funding Source: Supported by NIGMS 2R15GM078077‐02 and 3R15GM078077‐02S1
Molecular and cellular biology adaptive virtual learning environment (618.32)
Authors: Sawarynski Kara , Thomas David , Collins Brian , Weebadde Prabode
Published: June 2021

Molecular and cell biology is a constantly evolving area of science that encompasses many diagnostic as well as treatment plans within the medical field. Obtaining a robust ability to comprehend and analyze molecular medicine techniques is paramount for future physicians’ success within this rapidly developing arena. Due to several factors, hands‐on biology laboratories often are not present in a medical school curriculum. Although these topics are often covered in a lecture setting, the critical thinking and conceptual understanding of the molecular tools themselves are frequently only cursorily taught. We have developed an adaptable platform designed to help learners develop a deeper understanding of topics related to complicated, clinically relevant experiments and prepare them to be better able to transfer the knowledge to their future medical careers. Students will be required to make virtual experimental plan choices, which will each have an impact on their final result just as occurs in reality. To support independent learning, platform administrators scaffold learning with curated resource modules that are designed to support deep learning. The administration of an adaptable virtual learning environment will serve to enhance the understanding of complicated molecular medicine techniques, and promote the development of more capable physicians within the intricate molecular and biology field.
Stimulating 5th grade science students' interests through engaging hands‐on inquiry‐based lessons (618.37)
Authors: Booth Rachell , Banta Marilyn , Woodard Addie , Brown Susan , Matias Cindy , Maeder Corina
Published: June 2021

Students in the 5th grade at Hernandez Elementary School in the San Marcos Consolidated Independent School District (SMCISD) engaged in inquiry based hands‐on activities with their teachers led by faculty and students from the College of Science and Engineering at Texas State University. Two distinct events were used to increase the excitement for and conceptual understanding of concepts required for the 5th grade STAAR (State of Texas Assessment of Academic Readiness) test. First, fifty‐seven fifth‐grade students, four teachers, and four parents from Hernandez Elementary School attended a STEM field trip at Texas State University. Students rotated through a series of hands‐on activities, tours and demos spanning across chemistry, biology, physics, and computer science departments. Assessment of the field trip suggested that enthusiasm for science increased. Second, inquiry‐based activities were brought to the elementary school in a two‐week “science camp” in the spring. Junior level biochemistry students led the activities, which covered a range of concepts, including landforms, properties of water, and the food chain. Impact of the hands‐on experiments was assessed by monitoring the 5th grade STAAR test results. The findings indicated that the 5th grade students had performed better in not only science, but also reading and math.

Grant Funding Source: Supported by ASBMB HOPES Grant
Process Oriented Guided Inquiry Learning to enhance learning of concepts in a biochemistry lab (618.9)
Authors: Campbell Naomi
Published: June 2021

The objective of this study was to develop laboratory modules that incorporated Process Oriented Guided Inquiry Learning (POGIL) to enhance minority students' learning of important concepts in the biochemistry laboratory. The biochemistry laboratory was divided into two groups that met on different days. The exact same laboratory techniques were taught in both labs. The control group had the regular laboratory instruction and the test group recieved POGIL modules. The POGIL group scored significantly higher on the mid‐semester assessment exam than the control group.

Grant Funding Source: Supported by National Science Foundation DUE Award #0941955
Iron‐biofortified pearl millet improves attentional function in Indian adolescents, a 6‐month randomized controlled trial (619.2)
Authors: Scott Samuel , Murray‐Kolb Laura , Wenger Michael , Udipi Shobha , Ghugre Padmini , Haas Jere
Published: June 2021

Iron deficiency affects an estimated one in four persons globally and has been shown to impair cognition, though this relation in adolescents has not been substantiated. In a double‐blind efficacy trial, Indian adolescents (n=264) were randomized to groups consuming either high‐iron biofortified (Fe‐PM) or normal‐iron (CN‐PM) pearl millet. Here, we present results from a subset (n=161; 12‐16 y; 59 F) who performed tests of perceptual and cognitive functioning before and after 6 months of daily pearl millet consumption. Three speeded tests were administered: 1) Simple Reaction Time (SRT), a test of perceptual processing speed, 2) Go/No‐Go (GNG), a test of inhibitory control and 3) Attentional Network Task (ANT), a test of three critical functions of attention (low‐level attentional capture, high‐level attentional selection, and volitional control under distraction). In the intention‐to‐treat analyses, improvement was greater, though not statistically significant, in the Fe‐PM vs. the CN‐PM group for all three tests. In subgroup plausibility analyses, ferritin responders exhibited greater improvement (p<0.05) for the SRT and ANT vs. non‐responders. Hemoglobin responders improved more than non‐responders on the GNG (p<0.05) but not the SRT or ANT. These findings offer promise that iron treatment may improve specific aspects of perceptual and cognitive performance in adolescents.

Grant Funding Source: Supported by HarvestPlus/IFPRI
Comparison of mineral content in breast milk between early and later lactation periods among indigenous women from Western Highlands of Guatemala (623.11)
Authors: Li Chen , Wren Hilary , Xu Ran , Solomons Noel , Chomat Anne Marie , Scott Marilyn , Koski Kristine
Published: June 2021

Background: Human breast milk is the normal source of minerals for infant growth, development and metabolic function up to 6 months, but little information is available about how mineral concentrations vary throughout lactation in developing countries. Our objectives were to compare mineral concentrations between early (<45d) and later (4‐6mo) lactation periods, and to determine if mineral concentrations varied with infant age within these two lactation periods.

Methods: Inductively Coupled Plasma Mass Spectrometry was used to analyze the concentration of 13 minerals (Na, K, Ca, Mg, Mn, Zn, Cu, Cr, Sr, Se, Rb, Fe, P) in early (n=54) and later (n=49) breast milk samples collected from Mam‐Mayan women from rural villages in the Western Highlands of Guatemala.

Results: Median concentrations of Na, K, Mn, Zn, Cu, Cr, Rb and Fe were significantly higher in early milk compared with later milk whereas Mg was lower in early milk. No differences were observed between lactation period for Ca, Se and Sr. Within the first 45d, Na, K, Cu and Zn were negatively correlated with infant age. During the 4‐6mo interval, only Se was negatively correlated with infant age.

Conclusion: Our findings reveal that concentrations of Na, K, Cu and Zn decreased within the first 45d, that Mn, Cr, Rb and Fe had declined by 4‐6mo, and that Se declined during the 4‐6mo interval.

Grant Funding Source: McGill University International Mobility Award, NSERC
The synergistic effect of 1’‐acetoxychavicol acetate and sodium butyrate on the death of human hepatocellular carcinoma cells (644.17)
Authors: Matsui‐Yuasa Isao , Kato Rie , Azuma Hideki , Kojima‐Yuasa Akiko
Published: June 2021

It has been suggested that the combined effect of natural products may improve the effect of treatment against the proliferation of cancer cells. We evaluated the combination of 1’‐acetoxychavicol acetate (ACA), obtained from Alpinia galangal, and sodium butyrate (NaB), a major short chain fatty acid, on the growth of HepG2 cells and found that treatment had a synergistic inhibitory effect.

The number of HepG2 cells was synergistically decreased via apoptosis induction with the combined treatment of ACA and NaB. In ACA‐ and NaB‐treated cells, intracellular reactive oxygen species (ROS) levels and NADPH oxidase activities were increased significantly. The decrease in cell number after combined treatment of ACA and NaB was improved with the treatment of catalase. These results suggest that an increase in intracellular ROS levels is involved in cancer cell death. AMP‐activated protein kinase (AMPK) plays an essential role in controlling processes related to tumor development. In ACA‐ and NaB‐treated cells, AMPK phosphorylation was induced significantly, and this induction improved when cells were pretreated with catalase. These results suggest that the increase in intracellular ROS is involved in the increase of AMPK phosphorylation.

In conclusion, combined treatment with ACA and NaB synergistically induced apoptotic cell death via an increase in intracellular ROS and phosphorylation of AMPK.
Peptides in common bean (Phaseolus vulgaris L.) non‐digestible fraction inhibit human colorectal cancer cell survival in vitro through oxidative stress injury: a comparative study (644.5)
Authors: Luna‐Vital Diego , De Mejia Elvira , Dia Vermont , Loarca‐Pina Guadalupe
Published: June 2021

The aim was to characterize peptides present in non‐digestible fraction (NDF) of common bean cultivars, Azufrado Higuera (AH) and Bayo Madero (BM), produced after pepsin‐pancreatin digestion, determine their antiproliferative action on human colorectal cancer cells and their mechanism of action. Five peptides represented 70% of total protein in both cultivars (GLTSK, LSGNK, GEGSGA, MPACGSS and MTEEY). HCT116 colorectal cancer cell line was most sensitive to bean AH (IC50=0.53 mg/mL), and RKO to BM (IC50=0.51 mg/mL) peptide extracts. Cultivars AH and BM increased significantly the expression of p53 in HCT116 cells by 76% and 68%, respectively. AH modified the expression of cell cycle regulation proteins p21 (64%) and cyclin B1 (‐45%). BM modified the expression of apoptosis‐related proteins BAD (22%); cytC (66%), c‐casp3 (115%); Bax (50%), Bcl‐x (‐41%); Livin (‐32%); Survivin (‐27%) and TNFR1 (‐51%). Moreover, peptide extracts of both cultivars modified the expression of oxidative stress response‐related genes. In HCT116, NRF2‐related antioxidant enzymes were up‐regulated. Glutathione redox‐related enzymes were down‐regulated in RKO, potentially causing reactive oxygen species imbalance. In conclusion, peptides in common bean NDF contributed to the antiproliferative effect on human colorectal cancer cells by modifying molecules involved in cell cycle progression and apoptosis.

Grant Funding Source: Supported by University of Illinois‐University of Queretaro research grants
Acute effects of brewed cocoa consumption on sustained attention, motivation to perform work and feelings of anxiety, energy and fatigue (647.1)
Authors: Boolani Ali , Lindheimer Jacob , Loy Bryan , O'Connor Patrick
Published: June 2021

Acute effects of brewed cocoa and brewed cocoa with 49 mg added caffeine on attention, motivation and mood were examined in a randomized, double‐blinded, crossover trial. Participants (n=23) were low consumers of polyphenols without elevated feelings of energy. Prior to and three times after treatment, a 26‐minute battery was performed assessing motivation, mood and sustained attention (serial subtractions of 3 and 7, the continuous performance task and the Bakan dual task). Treatment X Time ANCOVAs controlling for prior night’s sleep showed that compared to placebo, cocoa reduced overall false alarms and speeded response times on the Bakan secondary task that was completed 31‐47 minutes post‐consumption. Caffeinated cocoa showed: (i) an attenuation of the anxiety provoking effects of cognitive testing found after drinking caffeine alone, and (ii) increased accuracy and reduced omission errors on the primary task of the Bakan test compared to cocoa alone. Brewed cocoa can acutely influence aspects of sustained attention but it has little effect on motivation to perform cognitive tasks or feelings of energy and fatigue. Supplemental caffeine in brewed cocoa can enhance aspects of sustained attention while brewed cocoa can attenuate the anxiety provoking effects found from drinking caffeine alone.

Grant Funding Source: The Hershey Company
Overexpression and function of small conductance Ca2+‐sensitive K+ channel (SKCa) isoform 3.1 in HL‐1 myocyte mitochondria (648.12)
Authors: Yang Meiying , Camara Amadou , Bienengraeber Martin , Kwok Wai‐Meng , Stowe David
Published: June 2021

SKCa agonists protect against cardiac ischemia and reperfusion injury. SKCa channels are present in cell membranes of vascular and cardiac atrial cells, but not in cell membranes of cardiac ventricular cells. Of the 3 known SKCa isoforms, we found SKCa isoform 3 of splice variant 1 (SKCa 3.1) in the inner mitochondrial (m) membrane of guinea pig ventricular cells. To determine its mitochondrial targeting sequence and function, we cloned full length (FL) and truncated SKCa 3.1 cDNA for overexpression in HL‐1 myocytes. SKCa 3.1 localization was examined by immunocytochemistry and western blotting and channel function was evaluated by K+ uptake into HL‐1 myocyte mitochondria with the K+‐selective fluorescent probe PBFI AM. We found that SK3FL‐EGFP, N‐terminus truncated SK3.1 (SK3Δ1‐277‐EGFP), but not C‐terminus (includes calmodulin binding motif) truncated SK3.1 (SK3Δ626‐720‐EGFP), localized into mitochondria when transfected into HL‐1 cells. Overexpression of SK3FL‐EGFP and SK3Δ1‐277‐EGFP, but not SK3Δ626‐720‐EGFP, increased the rate of K+ uptake in HL‐1 cell mitochondria in response to added KCl and CaCl2. K+ uptake by overexpressed SK3FL‐EGFP was sensitive to the SKCa blocker apamin. Our results show that mSKCa 3.1 requires its C‐terminus not only for mitochondrial targeting but also for Ca2+ ‐stimulated K+ uptake into mitochondria.

Grant Funding Source: R01 HL 089514
Novel murine model of hepatocyte‐specific human platelet‐derived growth factor alpha expression (649.9)
Authors: Kikuchi Alexander , Lee Jung Min , Monga Satdarshan
Published: June 2021

Hepatic fibrosis is a common outcome of chronic liver disease from many etiologies and represents a potentially reversible stage of disease progression. Hepatocytes play an important role in this pathologic process through apoptosis, and release of cytokines and growth factors to influence myofibroblast activation and altered proliferation. We have found elevated hepatocyte platelet‐derived growth factor α (PDGFRα) expression in experimental fibrosis and in patients with fibrosis, as well as reduced injury in PDGFRα‐conditional knockout mice. While these findings suggest that PDGFRα upregulation may be involved in hepatic injury, it remains undetermined whether PDGFRα upregulation in hepatocytes plays a compensatory or pathological role. To address this question, we have generated a novel transgenic mouse model expressing human PDGFRA in hepatocytes under the control of an albumin‐promoter. We show that this animal expresses human PDGFRα in whole liver lysates, and overexpresses total PDGFRα but lacks an overt phenotype. In conjunction with previously described human and mouse‐specific monoclonal anti‐PDGFRα antibodies, this mouse strain will allow the investigation of a cell‐specific role of PDGFRα in experimental models of chronic liver injury and regeneration.

Grant Funding Source: T32: HL094295 ANGIOPATHY TRAINING GRANT; R01: DK095498 ROLE OF PDGFRALPHA IN LIVER PATHO‐BIOLOGY
Acute high fat diet promotes NF‐κB activation and cardioprotection through circulating adiponectin (652.1)
Authors: Amratia Avni , Haar Lauren , Wani Mashhood , Gonzalez Christopher , Jones Walter
Published: June 2021

Prolonged high fat diet (HFD) consumption leads to diverse pathologies such as obesity, type II diabetes, atherosclerosis, and myocardial infarction. Paradoxically, studies have shown that short term HFD is cardioprotective against myocardial ischemia and reperfusion (I/R). This response is NF‐κB dependent, a hallmark of preconditioning pathways. This study will address the hypothesis that the adipose‐derived circulating cytokine, adiponectin promotes the activation of NF‐κB in an acute HFD by acting on cardiac receptors and stimulating cardioprotective gene programs.

To investigate this in an intact in vivo AdipoQ KO model, infarct size formed in 24h HFD and ctrl chow fed mice were compared. In vitro studies employed HL‐1 cells treated with adipocyte conditioned or ctrl medium to assess a direct response of adiponectin on cardiomyocytes after undergoing a simulated I/R (sim I/R) event in a hypoxic chamber. Cell death was measured by lactate dehydrogenase assay and NF‐κB activation was indirectly measured through the activation downstream NF‐κB ‐dependent genes (VEGF, HIF1a) by qRT‐PCR.

Results showed no significant difference in infarct size between the HFD and ctrl chow AdKO mice suggesting the loss of HFD cardioprotection due to loss of adiponectin. Furthermore, treatment in conditioned medium demonstrated significantly reduced cell death and upregulation of NF‐κB‐dependent gene programs after sim I/R in comparison to untreated cells. Our data supports adiponectin as a crucial molecular activator in acute HFD cardioprotection.
Investigation of the mechanism(s) of flavonol‐induced cardioprotection in rat isolated hearts (652.7)
Authors: CHIN KAI YEE , Darby Ian , Woodman Owen
Published: June 2021

The synthetic flavonol 3’,4’‐dihydroxyflavonol (DiOHF) reduces myocardial ischaemia/reperfusion (I/R) injury however the mechanism of DiOHF‐induced cardioprotection remains to be elucidated. We tested the hypothesis that the DiOHF‐induced cardioprotection results from selectively inhibiting injurious kinases without affecting protective kinases. Langendorff‐perfused rat hearts were subjected to 20 min global ischaemia followed by 30 min reperfusion in the presence of DiOHF (10 µM) or its vehicle (0.5% dimethyl sulfoxide). Hearts treated with DiOHF showed improved post‐ischaemic cardiac function and lower lactate dehydrogenase (LDH) release compared to its vehicle. DiOHF treatment significantly reduced I/R‐induced increased phosphorylation of c‐Jun N‐terminal kinases (JNK), measured by Western blot, while I/R‐activated phosphorylation of p38 mitogen‐activated protein kinase, extracellular signal‐regulated kinases (ERK) 1/2 and signal transducer and activator of transcription 3 were not affected. In addition, the phosphorylation of the multifunctional enzyme, Ca2+/calmodulin‐dependent kinase II (CaMKII) was elevated in I/R and was reduced by DiOHF. These data suggest that DiOHF‐induced cardioprotection against I/R injury is mediated by inhibiting the phosphorylation of the injurious kinases JNK and CaMKII without affecting protective kinases.
Tyrosine phosphorylation of HSC70 may mediate methotrexate resistance in murine L210 leukemia cells (655.5)
Authors: Liu Tuoen , Singh Ratan , Sheridan Peter , Rios Zechary , Bhushan Alok , Lai James , Daniels Christopher
Published: June 2021

We previously identified and characterized a 66‐68 kDa membrane‐associated, tyrosine phosphorylated protein in murine leukemia L1210 cells as HSC70 which is a methotrexate (MTX) ‐ binding protein. We further characterize the functional role of HSC70 in regulating MTX resistance in L1210 cells. The tyrosine phosphorylation status of the HSC70 in the membrane fraction is different between the parental L1210/0 and cisplatin (CDDP)‐MTX cross resistant L1210/DDP cells. The binding assay with MTX‐agarose beads showed HSC70 in the L1210/DDP cells had less binding ability with MTX compared with L1210/0 cells. In addition, genistein (a tyrosine phosphorylation inhibitor) significantly enhanced the resistance of L1210/0 cells to MTX. In addition, site‐directed mutation studies indicated the importance of tyrosine phosphorylation of HSC70 in regulating its binding to MTX. These studies suggested that tyrosine phosphorylation may contribute to the functions of HSC70 in regulating MTX resistance in L1210 cells.

Grant Funding Source: Supported by National Institutes of Health Grant P20 RR16454 and P20 GM103408
Ramalin suppresses adipocyte differentiation though the MAPKs pathway in 3T3‐L1 preadipocyte (657.10)
Authors: Lee Heeweon , Lee Chunggi , Jang Yeonjung , Pyo Suhkneung
Published: June 2021

Obesity is a main risk factor for several metabolic disorders such as type 2 diabetes, hypertension, and cardiovascular diseases. Obesity is also recognized as a low‐grade chronic inflammation. In the current study, the inhibitory effect of ramalin, isolated from Antarctic lichen, Ramalina terebrata, on adipocyte differentiation in 3T3‐L1 cells was examined. Ramalin attenuated the accumulation of lipid droplets stained with Oil red O in non‐toxic concentration. Western blot assay showed that ramalin remarkably inhibited the level of peroxisome proliferator‐activated receptor γ (PPAR γ) and CCAAT/enhancer‐binding protein β (c/EBP β) as well as CCAAT/enhancer‐binding protein α (c/EBP alpha) in a concentration dependent manner. Real‐time PCR assay revealed that the expression of MDI‐induced adipogenesis related genes (GLUT4, aP2, leptin) was significantly suppressed by ramalin. Our data also showed that ramalin significantly decreased phosphorylation of ERK1/2, p38 and JNK in a concentration‐dependent manner. Results from ELISA assay demonstrated that ramalin reduced the production of IL‐6 and TNF‐α. Taken together, ramalin can be a novel therapeutic candidate for controlling adipogenesis in preadipocytes.
Effects of selective dopamine D3 receptor compounds in nonhuman primate models of cocaine and methamphetamine abuse (661.8)
Authors: John William , Green Heather , Newman Amy , Nader Michael
Published: June 2021

Within the dopaminergic system, the D3R has been shown to mediate many of the behavioral effects of psychostimulants associated with high abuse potential. The present studies examined the effects of D3‐selective partial agonists and D3/D2R antagonists in three models of cocaine and MA abuse in rhesus monkeys. The highly selective D3 partial agonist PG619 and the D3/D2R antagonists buspirone and eticlopride were administered chronically to monkeys self‐administering cocaine and MA (n=3/group) under a food‐drug choice procedure in which complete cocaine and MA dose‐response curves were determined each session. Neither PG 619 (0.1‐3.0 mg/kg, i.v.), buspirone (0.01‐0.3 mg/kg, i.m.) nor eticlopride (0.001‐0.01 mg/kg, i.v.) significantly decreased cocaine or MA choice and intake while buspirone significantly increased the choice of low drug doses. The ability of buspirone to block reinstatement was also examined. Using the choice procedure in which saline was substituted for the self‐administered drug, buspirone (1.0‐1.7 mg/kg, p.o.) was found to block both cocaine‐ and MA‐induced reinstatement. In relation to this, buspirone (0.03‐0.3 mg/kg, i.m.) was also found to be effective in blocking cocaine and MA discrimination in a separate group of monkeys (n=3/group). Overall, these data indicate that while buspirone did not block the reinforcing effects of stimulants when studied under a food‐drug choice paradigm, it was effective in reducing reinstatement and in attenuating the discriminative stimulus effects of cocaine and MA, supporting a potential use for this compound in drug abuse treatment. DA012460

Grant Funding Source: Supported by DA012460
l‐Stepholidine is a potent pan‐dopamine receptor antagonist at G protein and β‐arrestin mediated signaling in vitro (662.5)
Authors: Meade Julie , Free R , Miller Nicole , Moritz Amy , Conroy Jennie , Doyle Trevor , Watts Val , Sibley David
Published: June 2021

Dopamine receptors (DARs) are highly validated drug targets in neuropsychiatric disease and, as such, compounds acting on this receptor family are both scientifically and clinically important. The Chinese herbal extract component l‐stepholidine has been reported to exhibit D2 DAR antagonism and D1 DAR agonist‐like activities in a number of in vivo behavioral analyses. Prior studies have further shown complicated mixed pharmacological responses involving both dopamine and serotonin receptors; however, clear in vitro characterization on individual DAR subtype signaling pathways has not been extensively explored. These data are particularly important given the recent discovery of the potential for signaling bias within the DAR family. In this study we present a broad examination of the biological actions and binding activities of l‐stepholidine on all DAR subtypes. l‐Stepholidine demonstrated relatively high binding affinity for all DAR subtypes, but failed to display any agonist response in either G protein‐ (cAMP assays and GTPγS binding), or β‐arrestin‐ (β‐arrestin translocation assays) mediated signaling at any DAR tested. Furthermore, l‐stepholidine was a full antagonist of dopamine mediated signaling at both D1‐ and D2‐like receptors and did not display signaling bias, thereby demonstrating that it acts as a non‐selective pan‐dopamine receptor antagonist in vitro.

Grant Funding Source: NIH Intramural Research Program
CHAC1 degrades glutathione, sensitizing cells to oxidative injury (663.10)
Authors: Crawford Rebecca , Higdon Ashlee , Prescott Eugenia , Mungrue Imran
Published: June 2021

CHAC1 is a pro‐apoptotic member of the endoplasmic reticulum stress pathway. The yeast homologue (YER163c) is the first known cytosolic protein to actively degrade glutathione (GSH), the most abundant antioxidant in the cell. However, the enzymatic activity of CHAC1 has not been fully characterized in mammalian cells. Herein, we investigate the effects of modulating CHAC1 in vitro on levels of cellular GSH and components of the GSH metabolic pathway in mammalian cells. Additionally, we define a role for CHAC1 in sensitizing cells to oxidative damage following heme treatment.

Overexpression of CHAC1 in human and mouse cells leads to a large decrease in total GSH, comparable to treatment with 250uM buthionine sulphoximine, a chemical inhibitor of GSH synthesis. We probed the effects of co‐administration of excess heme, an inducer of intracellular oxidative stress. CHAC1 overexpression increased sensitivity to heme‐mediated toxicity and decreased total GSH. Supplementation with N‐acetyl cysteine, a GSH precursor, alleviated this sensitivity. CHAC1 overexpression and knockdown leads to modulation of the mRNA for the GSH anabolic enzymes as measured by qPCR.

Collectively, this evidence suggests that CHAC1 sensitizes cells to oxidative stress due to degradation of GSH and potential feedback in the GSH anabolic pathway.

Grant Funding Source: RRC supported by Louisiana Board of Regents Fellowship #LESQSF; INM supported by NIH grant #HL094709
Essential role of adiponectin in ischemic postconditioning cardioprotection and its association with signal transducer and activator of transcription 3 (663.7)
Authors: Li Haobo , Irwin Michael , Xia Zhengyuan
Published: June 2021

Ischemic postconditioning (IPostC) protects the heart against ischemia reperfusion injury (IRI) by activating signal transducer and activator of transcription 3 (STAT3). Adiponectin (APN), a protein with anti‐IRI property, activates STAT3 (Cell Metab. 2011, 13: 401‐12), but its role in IPostC is unknown. We postulated that APN is essential for IPostC to confer cardioprotection. Wild type (WT) and APN knockout (KO) mice were subjected to 30 min coronary occlusion followed by 120 min reperfusion without or with IPostC achieved by 3 cycles of 10s of ischemia/10s of reperfusion. Cardiac function was assessed by pressure‐volume (PV) loop system. At the end of reperfusion, KO mice displayed larger infarct size and higher plasma creatine kinase‐MB, but lower end‐systolic PV relation (a reliable measure of ventricular function) and dP/dt (all P<0.05, vs. WT). IPostC significantly attenuated all these changes and increased phosphorylations of cardiac STAT3 (at Ser727), Akt (at Ser473) and eNOS in WT but not in KO mice. In cultured H9C2 cells exposed to 60 min hypoxia (H)/120 min reoxygenation (R), gene silencing with APN or STAT3 siRNA all abolished hypoxic postconditioning (HPostC, 3 cycles of 5 min H/5 min R) mediated cell protection. APN supplementation restored HPostC protection in APN siRNA treated but not in STAT3 siRNA treated cells. We conclude that APN is a key protein for IPostC to activate STAT3 to confer protection.

Grant Funding Source: Supported by RGC/GRF grant (784011) and NSFC grant (81270899)
Inflow versus outflow ischemia: differentiation and pharmacological characterization using cellular models (663.8)
Authors: Yang Ya , Lai Ted
Published: June 2021

Ischemia is a hallmark of stroke, myocardial infarction, and many other tissue injuries, and perhaps one of the best modeled human disease in laboratory animals. Nevertheless, it remains difficult to dissect the two main components of ischemia, the disruption of blood inflow versus blood outflow, in animal models. In this study, we attempt to differentiate these two components of tissue ischemia using cellular models. Inflow‐ischemia is induced by exchanging the nutrient‐enriched culture medium with a nutrient‐less extracellular solution, thereby mimicking the loss of nutritious blood supply. Outflow‐ischemia is induced by reducing the volume of the culture medium to allow more efficient accumulation of secretory products including metabolic wastes, thus resembling the disruption of blood outflow during tissue ischemia. Interestingly, while human embryonic kidney (HEK) cells are resistant to inflow‐ischemia, they degenerate abruptly upon outflow‐ischemia. In marked contrast, rat primary cortical neurons are highly susceptible to inflow‐ischemia, but strongly resistant to outflow‐ischemia. Lastly, the mouse endothelioma cells are resistant to either inflow‐ and outflow‐ischemia alone, and degenerated only when simultaneously subjected to both components of ischemia. As proof‐of‐concept, we demonstrated that outflow‐ischemia is transferrable between cell cultures by exchanging their culture media, and is fully reversible by re‐administration of fresh culture medium. Consistent with metabolic acidosis, cell death could be exacerbated and mitigated by decreasing and increasing pH of the culture medium, respectively. Moreover, the inhibitor amiloride prevented outflow‐ischemic death in a dose‐dependent manner. Taken together, the data reported here showed that different cell types are differentially susceptible to different components of ischemia, and further identified amiloride‐sensitive receptors as pharmacological targets against outflow‐ischemia.

Grant Funding Source: Supported by the National Science Council of Taiwan
Infusion of cardioplegia with monounsaturated lipids attenuates myocardial injury: involvement of lipid rafts in the protective effect (667.8)
Authors: Maldonado Claudio , Rodriguez‐Alvarez Walter , Khundmiri Syed , Pantalos George , Bauer Philip , Keeling W. Brent , Gray Laman , Perez‐Abadia Gustavo
Published: June 2021

Myocardial ischemia/reperfusion injury (IRI) can lead to deleterious clinical outcomes following cardiopulmonary bypass (CPB). We developed fusogenic lipid vesicles (FLVs) formulated with monounsaturated lipids (MULs) that modulate endothelial cell (EC) adhesion molecule expression. The purpose of this study was two fold: 1) determine in a swine CPB model the effect of infusing crystalloid cardioplegia (CC)+FLVs in attenuating IRI; and 2) elucidate potential mechanisms by which FLV administration provides protection. We hypothesize that FLVs rapidly incorporate MULs into EC lipid raft microdomains (LRMs) altering their composition, which in turn interferes with EC activation signaling. To evaluate efficacy of FLV treatment swine hearts were infused with CC or with CC+FLVs and underwent 2h of cross‐clamp ischemia. After clamps were removed, cardiac function was evaluated during 2h of reperfusion. To evaluate potential cellular mechanisms of FLV protection, in vitro studies to visualize incorporation of MULs into EC LRMs using advanced fluorescent microscopy techniques were performed by transfecting cells with plasmid expressing green fluorescent protein‐labeled flotillin‐1, and by treating cells with rhodamine‐labeled FLVs. Results from CPB studies indicated that CC+FLVs treatment reduced myocardial injury and improved cardiac stroke work index, and results from in vitro EC studies indicated that incorporation of FLV MULs into membranes was predominantly associated with LRMs. We conclude that FLV induced incorporation of MULs attenuates myocardial injury after CPB, and that the protective effect appears to be associated with the incorporation of MULs into EC LRMs.

Grant Funding Source: Supported in part by grants from NIH 1R42HL079855 and EndoProtech, Inc.
Recruitment of inflammatory cells to the bladder endothelium exposed to cigarette smoke extract (669.1)
Authors: Marentette John , McHowat Jane
Published: June 2021

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a debilitating disease of unknown etiology. Several risk factors are associated with IC/PBS including smoking, weight and diet. Platelet activating factor (PAF) plays a role in inflammatory cell recruitment to the endothelium and we have shown that PAF metabolism by PAF‐acetylhydrolase (PAF‐AH) is inhibited by cigarette smoke. We propose that cigarette smoking inhibits bladder endothelial PAF‐AH activity and contributes directly to inflammatory cell recruitment in the bladder wall. Incubation of human bladder microvascular endothelial cells (HBMEC) with cigarette smoke extract (CSE, 20 µg/mL) resulted in a significant inhibition of PAF‐AH activity with increased PAF accumulation and polymorphonuclear leukocyte (PMN) adherence. Pretreatment of HBMEC with (S)‐bromoenol lactone to inhibit calcium‐independent phospholipase A2β and PAF production prior to CSE incubation lead to a significant reduction in PMN adherence to the bladder endothelium. Similarly, pretreatment of PMN with PAF receptor antagonists (WEB 4086 or Ginkgo biloba) prior to CSE caused a significant reduction in PMN adherence. We propose that targeting endothelial cell PAF production or blocking the PAF receptor may represent novel therapies for management of IC/PBS patients, particularly smokers.

Grant Funding Source: Supported by a President's Research Fund, Saint Louis University grant
Drug carrier particle adhesion depends on bifurcation angle (675.1)
Authors: Lamberti Giuseppina , Smith Ashley , Kiani Mohammad , Prabhakarpandian Balabhaskar , Pant Kapil
Published: June 2021

In this study, Idealized Microvascular Networks (IMN) representing different angles observed in the microcirculation were used to investigate the effect of bifurcation angle on drug carrier particle interaction with the vessel wall. IMN was fabricated using soft lithography processes followed by study of adhesion of biotin‐conjugated microspheres (diameter 2 µm) to avidin coated vessel walls. The networks used in these studies comprised bifurcation angles of 30°, 60°, 90°, and 120°.

Our findings indicate that particle adhesion decreased with increase in shear rate in both bifurcations and linear areas of the channels. However, bifurcations were found to have significantly higher particle adhesion (1.5X or more) compared to linear channels. Furthermore, particle adhesion was found to increases linearly with an increase in the bifurcation angle.

Our results show that drug carrier particle adhesion is significantly affected by the presence of bifurcations and their corresponding angle and therefore their design needs to be tailored to account for higher binding propensity at larger bifurcation angles.