bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of reactive oxygen species in L6 myoblasts by glucose transporter 1 (693.5)
Authors: Andrisse Stanley , Koehler Rikki , Chen Joseph , Patel Gaytri , Vallurupalli Vivek , Ratliff Benjamin , Warren Daniel , Fisher Jonathan 
Published: June 2021

Elevated reactive oxygen species (ROS) levels are associated with metabolic abnormalities such as insulin resistance. Glucose transporter 1 (GLUT1) transports glucose and dehydroascorbic acid, both of which play roles in antioxidant defense. We hypothesized that conditions that would increase GLUT1 activity would result in decreased ROS levels and conditions that would decrease GLUT1 activity would increase ROS levels. . L6 myoblasts loaded with H2‐DCFDA, a fluorescent ROS probe, were treated under various conditions to alter GLUT1 activity, and fluorescence readings were taken under basal conditions or after increases in ROS were induced by exposure to pyrogallol or antimycin A. GLUT1 inhibitors, fasentin and phloretin, increased induced ROS levels. Ataxia telangiectasia mutated inhibitors and activators, previously shown to inhibit and augment GLUT1 activity, increased and decreased basal and induced ROS levels, respectively. GLUT1‐S490A, previously shown to decrease GLUT1 activity, enhanced basal and induced ROS levels. GLUT1‐S490D, previously shown to increase GLUT1‐mediated transport, lowered basal ROS levels. Thus, the data suggest that GLUT1 plays a role in regulation of ROS levels.Grant Funding Source: Supported by: William Townsend Porter Predoctoral Fellowship from the American Physiological Society
PTK6 mediates intestinal endothelial barrier dysfunction in response to TNFα (695.1)
Authors: Haines Ricci , Beard Richard , Yuan Sarah , Wu Mack 
Published: June 2021

A key event in the progression of systemic inflammation resulting from severe trauma or shock involves hyperpermeability of the intestinal endothelium. The signaling events leading to increased endothelial permeability in these disease states are incompletely understood. Although the role of protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK) has been primarily studied in mechanisms underlying metastasis and cancer, it has also been shown that PTK6 participates in mediating colonic epithelial barrier dysfunction. In this study, we hypothesized that PTK6 is 1‐ expressed in intestinal endothelial cells, and 2‐ contributes to endothelial hyperpermeability in response to inflammation. To this end, we investigated the role of PTK6 in mediating the disruption of intestinal endothelial barrier function. Results showed that PTK6 was detected in intestinal endothelial cells at the level of protein and mRNA. In addition, PTK6 knockdown attenuated decrease in transendothelial electric resistance (TER) as measured by electric cell‐substrate impedance sensing (ECIS) of intestinal endothelial monolayers in response to inflammatory factors including TNFα. Furthermore, we showed that TNFα increased activity of PTK6 as evidenced by increased phosphorylation at Y342, supporting the hypothesis that this kinase plays a role in endothelial inflammation. We further studied the potential targets of PTK6 including junctional structure proteins to determine the molecular mechanism of its role in regulating endothelial barrier function. Our study explores a potential implication that targeting PTK6 may serve as a novel therapeutic strategy in treating diseases characterized by hyperpermeability of the intestinal endothelium.Grant Funding Source: Supported by VA Merit Review 5101BX000799, the NIH grants HL96640, HL61507 and HL70752
Activation of transient receptor potential vanilloid 1 decreases endothelial nitric oxide synthase phosphorylation at Thr497 by protein phosphatase 2B‐dependent dephosphorylation of protein kinase C (696.1)
Authors: Su Kuo‐Hui , Lin Shing‐Jong , Shyue Song‐Kun , Lee Tzong‐Shyuan 
Published: June 2021

We investigated the effects and underlying molecular mechanism of transient receptor potential vanilloid 1 (TRPV1), a calcium‐permeable non‐selective cation channel, on phosphorylation of endothelial nitric oxide synthase (eNOS) at threonine 497 (Thr497) in bovine aortic endothelial cells (BAECs) and in mice. In BAECs, treatment with the TRPV1 ligand evodiamine decreased the phosphorylation of eNOS at Thr497, protein kinase Cα (PKCα at Serine 657, and PKCβ2 at Serine 660. Evodiamine increased protein phosphatase 2B (PP2B) activity and promoted the formation of a PP2B‐PKC complex. Inhibition of TRPV1 activation, removal of extracellular calcium or pharmacological inhibition of PI3K/Akt/CaMKII/AMPK signaling pathway abolished the evodiamine‐induced alterations in phosphorylation of eNOS at Thr497, PKCα, PKCβ2 and PP2B activity, as well as the formation of a PP2B‐PKC complex. Inhibition of PP2B activation partially reduced the evodiamine‐induced NO bioavailability and tube formation in ECs and angiogenesis in mice. Moreover, evodiamine decreased the phosphorylation of eNOS at Thr497, PKCα, and PKCβ2 in apolipoprotein E (ApoE)‐deficient mouse aortas but not TRPV1‐deficient or ApoE/TRPV1 double‐knockout mice. TRPV1 activation in ECs may elicit a calcium‐dependent effect on PP2B‐PKC signaling, which leads to dephosphorylation of eNOS at Thr497 in ECs and in mice.Grant Funding Source: National Science Council, Taiwan
Evaluation of the scapula module from the interactive atlas of the human skeleton (725.10)
Authors: Casey Gregory , Cork R. 
Published: June 2021

Last year we developed a three component module of the scapula which included a short instructional video, an interactive animation, and a self‐testing quiz. This semester undergraduate nursing students were provided with access to this module in Block 3 (upper extremity) of their anatomy course. In order to evaluate its effectiveness we compared the scores on this semester’s Block 3 exam with the scores last Fall when they did not have access to the scapula module. Both groups of students were asked the same 11 scapula‐related questions. These questions were divided between the practical exam (6) and the written exam (5) and ranged from identifying bony landmarks to knowing the innervation of muscles attached to the scapula.In addition to analysis of their exam performance we also surveyed the students about their usage of the scapula module and their opinions of its usefulness. Overall, the scapula module received very positive reviews from the students with the interactive animations being the most popular feature. Most negative reviews related to technical issues arising from Browser differences and the inability of some features to work properly on tablets such as the iPad. Statistical analysis of the evaluation data is ongoing, but it does show a small but consistent improvement. The students who had access to the online scapula module did better on the scapula‐related questions than those who did not.
Three‐dimensional modeling of facial nerve VII (726.3)
Authors: Peterson Dani 
Published: June 2021

Due to its long and complicated trajectory through the cranium, facial nerve VII (CN VII) can be damaged in surgeries, sometimes resulting in facial muscle paralysis. We created a three‐dimensional (3D) model based on data from dissection of the nerve in a human cadaver in order to give physicians a greater in vivo knowledge of the pathway of CN VII. We dissected the lateral side of the right half of the head to the level of the parotid gland, identified the parotid plexus of CN VII, and followed its five branches. In addition, we are currently following the nerve through the internal auditory meatus on its pathway through the temporal bone. We imaged the head using Magnetic Resonance Imaging (MRI). The resulting images served as the anatomical template for the reconstructed nerve model. After completing the dissection, we tracked the nerve trajectory using a MicroScribe 3D Digitizer in the context of other nearby structures. The resulting map will aid surgeons by increasing accuracy when performing removals of acoustic neuromas and parotid tumors and in repairing the temporomandibular joint.Grant Funding Source: Supported by Mentoring Research Grant, College of Life Sciences, Brigham Young University
Comparative study of in vitro expanded somatic stem cells from different sources (732.3)
Authors: Danisovic Lubos , Bohac Martin , Novakova Zuzana , Varga Ivan , Bohmer Daniel , Polak Stefan 
Published: June 2021

Mesenchymal stem cells (MSCs) represent a unique tool of regenerative medicine. They are undifferentiated cells with the ability of long‐term self‐renewing and plasticity. The main goal of this study was biological and morphological analysis of human MSCs derived from bone marrow, adipose tissue and dermis in respect to cartilage tissue engineering. MSCs were isolated and cultured in vitro using standard protocols. They were maintained in a‐MEM supplemented by 15% of fetal bovine serum and antibiotics. MSCs from all tissues were cultured up to the 5th passage. The kinetics of proliferation was analyzed by CEDEX XS and expression of selected markers was assessed by flow cytometry. They were morphologically analyzed by inverted microscope and TEM. Pellet cultures were used to induce chondrogenic differentiation and this event was proved by real‐time PCR. MSCs from all sources were attached on the Petri dishes after 24 h and started to proliferate in colonies. After 7‐10 days they reached 80% confluency and were passaged. In the next 5 passages they displayed fibroblastoid morphology. MSCs also showed similar kinetics of proliferation and shared the expression of CD29, CD44, CD73, CD90, CD105 and CD166 and were negative for CD14, CD34, CD45 and HLA‐DR. TEM showed similar morphology of all analyzed MSCs. We have demonstrated that MSCs from bone marrow and adipose had potential to undergo chondrogenic differentiation comparable with chondrocytes, but this potential was decreased in dermis‐derived MSCs. We can emphasize that MSCs isolated from bone marrow, adipose tissue and dermis possess similar biological and morphological properties, but differ in chondrogenic potential. Dermis‐derived MSCs have an inferior potential for chondrogenic differentiation. So this fact decreases their potential for cartilage tissue engineering. Hoverer, further studies focused on applying of appropriate exogenous factors must be carried out to clarify this issue.Grant Funding Source: Supported by MZSR no. 2012/4‐UKBA‐4 and APVV no. 0434‐12.
Tumor suppressor properties and a role in maintaining genome integrity for the Meig1 gene (736.3)
Authors: Don Jeremy , Marciano‐Levi Iris 
Published: June 2021

Meig1 is a highly conserved gene first identified in mouse pachytene spermatocytes undergoing meiotic recombination. In these cells the MEIG1 protein enters the nucleus and forms foci along the meiotic chromosomes, reminiscent of foci obtain by proteins known to be involved in the DNA damage response. Meig1 knockout (KO) male mice are infertile. These mice exhibit increased apoptosis of post recombination spermatogenic cells and significantly fragmented DNA in the few epididymal sperm cells that could be obtained. Given these findings the objectives of this study were to examine the hypothesis that MEIG1 plays a role in the system responsible for maintaining genome integrity. Immunocytochemistry analysis of γH2AX signaling in meiotic chromosomes of late pachytene spermatocytes showed that in KO mice they were highly “decorated” with γH2AX foci, compared to WT chromosomes. Moreover, analysis of post‐recombination meiotic chromosomes revealed the existence of numerous chromosomal fragments in KO meiotic preparations whereas no such fragments could be seen in preparations from WT spermatocytes. Regarding somatic cells, KO MEFs exposed to etoposide treatment exhibited significantly slower disappearance of the γH2AX foci compare to WT MEFs, suggesting delayed or aberrant repair. Interestingly, most Meig1 KO mice (more than 30 mice to date), and about one third of the Meig1 +/‐ heterozygotes developed huge and aggressive tumors between 6‐18 months of age, whereas none of the age‐matched wild‐type (WT) siblings developed tumors. These tumors were identified as being mainly malignant adeno‐carcinomas and lymphomas. These results imply that Meig1 has tumor suppressive properties and suggest that it is involved in maintaining chromatin integrity not only in spermatogenic cells but in somatic cells as well, rendering it as a new player in the general DNA damage response, especially the response to DSB.This study was supported by grant No. 20072019B from the Israel Cancer Association.
Role of protein prenyltransferases in modifying effectors from infectious bacterium (739.7)
Authors: Wright Elia , Fierke Carol 
Published: June 2021

Protein prenylation is an important post‐translational modification. Many G‐proteins, such as Ras and Rho, are prenylated in order to anchor them to the cell membrane. Protein farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) add 15‐carbon farnesyl groups and 20‐carbon geranylgeranyl groups to proteins, respectively. Substrate specificity for FTase and GGTase is minimally described by the Cza2X paradigm where C is a cysteine residue four amino acids from the C‐terminus, followed by two amino acids (z is variable; a2 is aliphatic) and an X group that specifies which prenyltransferase is preferred, though there is significant substrate overlap. Recent experiments have demonstrated that infectious bacterium Legionella pneumophila requires host cell farnesylation machinery to induce pulmonary disease. Ankyrin b, an essential Legionella effector protein, is farnesylated by the host FTase, which is important for evasion of the endocytic pathway and protein function. Prenylation of effector proteins may be a general method used by pathogenic bacteria to anchor proteins in hosts. To investigate the prevalence of prenylation of bacterial proteins, we have measured the reactivity of mammalian FTase and GGTase‐I both in vivo and in vitro with peptide sequences derived from proteins expressed in pathogenic bacteria. We have shown that FTase and GGTase are capable of prenylating bacterial Cza2X motifs in vitro and in vivo through peptide assays and in vivo cellular localization of GFP fusion proteins. We have also tested a series of prenyl donor analogs synthesized in the Gibbs laboratory with FTase and GGTase to identify analogs with similar substrate specificity to in vivo prenyl donors to use as tools for defining the prenylome. These studies will further our understanding of the many ways protein prenyltransferases regulate cellular processes and how inhibition of these enzymes may be incorporated into treatment of bacterial infections and other diseases.Grant Funding Source: Supported by NIH F31 Diversity Fellowship
Mechanisms of atypical protein kinase C activation in insulin signaling (767.9)
Authors: Tobias Irene , King Charles , Newton Alexandra 
Published: June 2021

Atypical protein kinase C (aPKC) is a key modulator of insulin signaling that regulates glucose transport. Dysfunction of aPKC correlates with insulin‐resistant states in both mice and humans. Despite the engaged interest in this enzyme’s importance to type 2 diabetes, much less is known about the molecular mechanisms that govern its cellular functions than for the conventional and novel classes of PKCs. Here we examine the regulation of aPKC by phosphorylation and lipid binding in order to better define these mechanisms of activation in the context of insulin signaling pathways. Specifically, we show that key drivers of aPKC activity are 1) constitutive phosphorylation at the activation loop and turn motif phosphorylation sites and 2) that phospholipids regulate activity and susceptibility to dephosphorylation. Methods include in vitro kinase activity assays performed on recombinant aPKC purified from insect cells, using phospho‐resistant or phospho‐mimetic mutations placed at key phosphorylation sites. Additional biochemical activity studies were performed following dephosphorylation and in the presence of phospholipids. Our data are consistent with aPKCs sharing similar constitutive phosphorylation as conventional and novel PKCs, with dephosphorylation and activity accelerated by the presence of lipids.Grant Funding Source: This work was supported by P01 DK054441 to ACN and T32 GM007752 to IT
Group III phospholipase A2: collective unity amidst structural and functional diversity (769.1)
Authors: Hariprasad Gururao , Singh Reema 
Published: June 2021

Secretory phospholipase A2 (PLA2) catalyses the hydrolysis of the sn‐2 position of glycerophospholipids to liberate arachidonic acid, a precursor of eicosanoids, that mediate inflammation. The group III PLA2 enzymes are present in a wide array of organisms across many species with completely different functions. For a systematic classification of the group, fifty protein sequences from different species of the group were taken for sequence, structural and phylogenetic studies. In addition to the conservation of calcium binding motif and the catalytic histidine, the sequences exhibit specific ‘amino acid signatures’. Structural modeling reveals that these enzymes have a conserved globular structure with species specific variations seen at the active site, calcium binding loop, hydrophobic channel, the C‐terminal domain and the quaternary conformational state. Phylogenetic analyses are in accordance with the structural features. The results provide a convincing platform for the classification the group III PLA2s into‐ 1A: venomous insects; IB: non‐venomous insects; II: mammals; IIIA: gila monsters; IIIB: reptiles, amphibians, fishes, sea anemones, liver fluke; IV: scorpions. This classification helps to understand structure‐function relationship, substrate specificity, designing of potent inhibitors against the drug target isoforms and understand evolutionary perspective of constituents.Grant Funding Source: Supported by FAST TRACK grant No. SR/FT/18 given by Department of Science and Technology
Exploring enzyme conformational landscape with pressure‐perturbation: allosteric rearrangements in P450 3A4 revealed with FRET and SDSL‐EPR (769.16)
Authors: Davydov Dmitri , Yang Znongyu , Halpert James , Hubbell Wayne 
Published: June 2021

We used a combination of pressure‐perturbation techniques to explore conformational rearrangements in cytochrome P450 3A4. Site‐directed incorporation of a FRET donor‐acceptor pair allowed us to discover an equilibrium between open and closed states revealed in pressure‐induced increase in the distance between fluorophores placed in the α‐ and β‐domains. This transition is characterized by a volume change (ΔV°) of ‐33 ± 14 ml/mol and ΔG° of 0.7 ± 0.2 kcal/mol, so that only 20% of the enzyme adopts the open conformation at ambient pressure. Addition of testosterone, an allosteric activator, changes the ΔV° and ΔG° to ‐18 ± 7 ml/mol and 0.2 ± 0.1 kcal/mol respectively, increasing the fraction of the open state to 40%. The studies by site‐directed spin labeling (SDSL) and pressure‐perturbation EPR demonstrated that this transition is also revealed in pressure‐dependent alteration of the mobility of a nitroxide probe placed at positions 85, 166, 374, 409, or 495. We also detected a pressure‐induced displacement of emission maxima of environment‐sensitive fluorophores placed at residues 85 and 409 suggesting their relocation to a more polar environment. Importantly, the parameters of these testosterone‐dependent changes were similar to those found in our FRET studies. Our results elucidate the mechanism of effector‐dependent conformational equilibrium in P450 3A4 and demonstrate a high potential of pressure‐perturbation strategies in the studies of enzyme conformational landscapes.Grant Funding Source: Supported by NIH GM54995
Time‐course analysis of gene expression in a mouse model simulating aspects of human post‐traumatic stress disorder (776.7)
Authors: Muhie seid , Srinivasan Seshamalini , Gautam Aarti , Chakraborty Nabarun , Hammamieh Rasha , Jett Marti 
Published: June 2021

We profiled transcriptome changes in brain regions and blood of socially defeated mouse model of post traumatic stress disorder. Brain regions involved in fear circuitry and blood were collected from 5 days and 10 days aggressor exposed and control mice. Changes in gene expression after 1 day, 10 days and 42 days of rest (post 5 days and 10 days defeat periods) were profiled using microarray and real time PCR. Differentially expressed blood transcripts were validated by assaying the corresponding plasma proteins.Functional and pathway analyses of differentially regulated genes in trauma response brain regions (Aygdala, hippocampus, medial prefrontal cortex, ventral striatum and nucleus acumbens) of defeated mice were found to be involved in startle response, associative learning, long term memory, inflammation, regulation of circadian rhythm, glucocorticoid receptor activity, dendritic branching, and synaptic depression.Blood transcripts with increased expression with longer rest period were involved in T‐cell immunity, serotonorgic and dopaminergic synaptic processes and axonal guidance. Transcripts in blood with decreased expression with longer rest period were involved in associative learning, opoid signaling and glutamatergic pathways. Changes at the transcriptome level indicated the pervasive nature of traumatic stress in altering functions and pathways which normally precipitate in phenotypic behavioral disorders and pathologies. With increased rest period, some of the important functions and pathways showed regaining vitality indicating a sense of recovery with time.
Electron paramagnetic resonance characterization of drug‐binding to monoacylglycerol lipase using the dynamic signature of a protein spin‐label (779.3)
Authors: Wong Brian , Budil David , Karaogeorgos Ioannis , Tyukhtenko Sergiy , Makriyannis Alexandros 
Published: June 2021

In recent years, monoacylglycerol lipase (MGL) has been an important pharmaceutical target because of its relationship to pathways that have been implicated in cancer proliferation. The purpose of this study is to find an efficient and streamlined way of characterizing drug‐binding to MGL using electron paramagnetic resonance (EPR). The free nitroxide radical methyl thiosulfonyl spin label (MTSL) was used to label the single cysteine mutant S208C on a domain of MGL that is thought to constitute a “lid” covering the substrate binding pocket of MGL. EPR spectra shows reproducible line shape changes reflecting a significant reduction in the mobility of the lid when MGL binds reversible inhibitors. The results are consistent with NMR data that reveal an equilibrium between the putative “open” and “closed” positions of the lid as a function of temperature or pH. These findings establish a useful assay for screening potential cancer drugs that bind in a specific way to MGL that can be carried out more rapidly and with significantly smaller amounts of protein than other assays for the dynamic motion of the drug target.Grant Funding Source: Supported by grants DA09158, DA00493, DA03801 and DA07312