bluuebunny/crossref_metadata_2025_split huggingface dataset:
Extracellular Superoxide Dismutase (SOD3) mediates extracellular superoxide‐dependent signaling by TNFalpha.
Authors: Miller Michael Raymond , Choi Hyehun , Lamb Fred S. 
Published: June 2021

Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O2−•) to be produced extracellularly by NADPH oxidase 1 (Nox1). It remains unclear how this redox signal is transduced into activation of intracellular signaling pathways. We sought to identify steps in the TNFα signaling cascade that are directly O2−•‐dependent. We analyzed TNFα receptor endocytosis and activation of MAPKs and NF‐κB in cultured vascular smooth muscle cells (VSMC). JNK, p38 and ERK1/2 were activated with a similar concentration dependence. TNFα receptor endocytosis, MAPKs (p‐JNK and p‐ERK1/2) and NF‐κB activation were all inhibited by unmodified superoxide dismutase (SOD, 500 U/ml) added extracellularly, but not by catalase (1000 U/ml), or by overexpression of cytoplasmic CuZnSOD or catalase with adenovirus. This suggested a paramount importance of extracellular O2−•. We next sought to determine if endogenous, extracellular SOD (SOD3) modulated these responses by employing siRNA‐mediated downregulation. Surprisingly, siSOD3 blocked TNFα‐induced endocytosis (37 % reduction) and rather than enhancing MAPK and NF‐κB activation, these effects were blocked (Relative NF‐κB activation; siControl+TNFα 10.8 ± 1.0, siSOD3+TNFα 4.0 ± 0.3 fold, n=7, p<0.05). Similar results were obtained by pharmacologic inhibition of SOD3 using DETC (100 μM). These results demonstrate that enzymatic activity of endogenous SOD3 is required for signaling, however, the lack of effect of extracellular catalase calls into question the role of H2O2, the product of SOD3 activity. In order to directly assess the signaling impact of H2O2 we performed a dose‐response to this oxidant. MAPK activation occurred at a lower concentration (1 μM) while a higher concentration (10 μM) potently inhibited signaling (Figure 1). It remains to be determined how SOD3 modulates local H2O2 concentrations in the extracellular matrix following Nox1 activation by TNFα, and whether the H2O2 produced by SOD3 is accessible to catalase placed into that compartment. Taken together, these data suggest a novel role for SOD3 in transducing Nox1‐dependent inflammatory signaling activated by TNFα in VSMC.Support or Funding InformationThis work was supported by R01 HL128386 (FSL) and by a Scientist Development Award from the AHA (HC).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Academic success predictors in Optometry
Authors: Foutch Brian , Trevino Richard , Fortepiani Lourdes 
Published: June 2021

The success of students in health professions training programs depends on the selection process, teaching and curriculum, motivation, and student support services. However, most of the factors considered as predictors of student success in medicine and nursing include those involved exclusively in the selection process, such as grade point average (GPA), standardized entrance and aptitude test results. Similar factors have been found to predict the GPA and graduation of students in optometry programs. However, we are unaware of any systematic evaluation in an optometry training program of student aspects beyond selection factors.In this study, we propose to widen the scope of the factors involved in academic success in optometry school to include demographic aspects (like gender and race), psychological aspects (like learning style), and exam platform. We also examined the classic academic aspects like GPA and standardized entrance test (Optometry Admission Test [OAT]) scores. As the fourth year in our curriculum is exclusively clinic and includes rotations outside of the optometry school, we chose GPA at the end of their in‐house curriculum as the index measure of academic success. To this end, we recruited sixty‐five students from the second‐year class at the University of Incarnate Word Rosenberg School of Optometry and recorded the following: scores from two exams in two concomitant pathophysiological courses during their third semester in the in‐house curriculum, demographic data (gender, race, and ethnicity), undergraduate GPA, OAT scores, and optometry GPAs at the mid‐point and at the end of their in‐house curriculum. We also determined learning styles via surveys.We found that race and ethnicity were predictors of student success at the mid‐point but not at the end of the in‐house curriculum. The effect of undergraduate GPA and OAT scores was negligible. However, student performance in the pathophysiological courses during their third semester and the GPA at mid‐point of their in‐house curriculum predicted academic success (r=0.662, p<0.001; r= 0.659, p<0.001, respectively). Apart from academic factors, learning style—specifically the “read‐write” index—significantly and positively correlated with academic success (r=0.328, p<0.08). These data suggest that additional attention to student success during the first half of the in‐house curriculum may be worthwhile and bolster academic success overall. In addition, it may be useful for optometry faculty to have knowledge of student learning styles, as students' preferred method of learning played a role in their success.Support or Funding InformationAmerican Optometric Foundation (AOF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palatal Epithelial Cells Migrate Independently of Ephrin Signaling during Fusion
Authors: Logan Shaun , Benson Douglas 
Published: June 2021

The mammalian secondary palate forms from two shelves of mesenchyme sheathed in a single layered epithelium. Failure of the midline epithelial seam (MES) to degrade when these shelves meet at midline prevents mesenchymal confluence and results in a cleft palate. Our previous studies showed that cultured MES cells undergo features of epithelial‐to‐mesenchymal transition (EMT) when ephrin reverse signaling is activated. We therefore hypothesized that midline epithelial cells are removed during fusion by migration away from the seam in response to ephrin action. In order to observe this migration, we performed real time imaging of fusing embryonic mouse palates in culture using two‐photon confocal microscopy. The palates came from mice expressing an ephrin‐B2‐driven green fluorescent protein (GFP) that specifically labels the nuclei of epithelial cells in the palate. Immediately after 48 h of imaging, tissues were fixed, sectioned, and examined for fusion. The motion of individual MES cells was tracked over the time course of culture using Imaris software. MES cells migrated in a surprisingly unidirectional fashion toward the oral surface over 48 h of imaging, regardless of tissue orientation on the culture dish, and maintained their GFP expression for the duration of the experiment. Fusion of the imaged palates was confirmed histologically, thus demonstrating that our culture time frame encompassed the period required for palatal fusion. Addition of unclustered EphA4/Fc recombinant protein during the culture period to block ephrin reverse signaling inhibited fusion, as we previously established, but did not change the observed migration of GFP‐labeled cells. Because these cells maintained GFP expression, we conclude that they remained alive and that MES cell death by apoptosis is therefore not required for fusion, as was previously thought. Although ephrin reverse signaling is required for palate fusion, we found that it is not necessary for MES cell migration.Support or Funding InformationNIH/NIDCR R01DE022804This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pelvic limb and tail musculature of the red kangaroo (Macropus rufus)
Authors: Payne Arielle , Schmalz Naomi , Byram Jessica , McNulty Margaret , Organ Jason 
Published: June 2021

Most members of the marsupial mammalian family Macropodidae move around in their environments using a bipedal form of hopping and are instantly recognizable by their elongated lower limbs and long, robust, nonprehensile tails. Paleontologists rely on anatomical descriptions of living species to make inferences about locomotor evolution and how anatomical specializations in kangaroos vary with body size. Even though the locomotor anatomy of the smaller eastern grey kangaroo (Macropus giganteus) is well documented, a review of the literature demonstrates a near absence of documentation of the musculoskeletal anatomy of the red kangaroo (Macropus rufus), the largest living marsupial. To fill this gap in knowledge, this work describes the musculoskeletal anatomy of the pelvic limb and tail of the red kangaroo and documents anatomical differences from the eastern grey kangaroo and other large terrestrial marsupials. We performed two complete pelvic limb and tail dissections on two lightly embalmed red kangaroos: one male adult obtained from Grant's Farm nature park (St. Louis, MO), and one juvenile male obtained from Louisiana State University School of Veterinary Medicine. Anatomical features and attachments of all muscles were recorded and photographed during the dissection; skeletal remains were macerated and cleaned after dissection by a dermestid beetle colony. Our studies of the pelvic and tail musculature of red kangaroos demonstrated differences between eastern grey and red kangaroos in attachments for two specific muscles of the proximal pelvic limb: The attachments of the ischiofemoralis in the red kangaroo differ in that this muscle's proximal and distal attachments form broad aponeuroses, as opposed to narrower tendinous attachments in the eastern kangaroo. The small lateral rotators of the hip (obturator internus, obturator externus, piriformis, and the two gemellus muscles) in the red kangaroo are more distinct from one another, and the distal attachment of the piriformis is distal to the greater trochanter of the femur instead of medial to it, as described in the literature for the eastern grey kangaroo. Additionally, prominent muscle bellies for three muscles originating in the distal pelvic limb and attaching to the pes are nonexistent or inconsistent with the literature: the extensor digiti II and III longus, peroneus digiti IV, and peroneus digiti V are not found in either red kangaroo specimen, even though they've been described for the eastern red kangaroo. Ultimately, more specimens are necessary to verify these differences and address inconsistencies in the literature before we can make any inferences about how these differences may subtly affect locomotion in these closely related taxa.Support or Funding InformationThis research was supported by Indiana University School of Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Role of RNA N6‐methyladenosine methylation in endothelial response to flow”
Authors: Chien Chian Shiu , Song Hui‐Yung , Chiou Shih‐Hwa , Chien Shu 
Published: June 2021

N6‐methyladenosine (m6A) has the most abundant modification among eukaryotic mRNAs, and plays a vital role in cellular responses to many types of physiological stress. Endothelial cells (ECs) are constantly exposed in the shear stress induced by blood flow over a long period of time. Oscillatory shear (OS) can cause inflammatory response and induce altherosclerotic plaque formation. In contrast, pulsatile shear (PS) has anti‐inflammatory effect and is atheroprotective. However, the relationship between RNA modification and the effects of shear stress in endothelial cells is still unclear. In time‐series experiments on endothelial cells subjected to pulsatile and oscillatory shears, we determined m6A modifications in purified mRNA in by RNA dot blot and the expression levels of regulators of m6A by RT‐PCR and western blot. The results show that the changes of m6A modification in endothelial cells in response to different shear stresses are dynamic events and that the m6A modification of mRNA is lesser in response to OS than PS. The expression levels of m6A writers Mettl3 and Mettl14 were up‐regulated by pulsatile shear as compared with oscillatory shear and static state. In summary, this study has shown that different shear stresses can induce distinct changes of m6A modification and that Mettl3 and Mettl14 were induced by pulsatile force and may regulate RNA N6‐methyladenosine methylation in endothelial response to flow.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitotic Inheritance of mRNAs encoding Transcription factors in Osteoblastic Cells.
Authors: Khani Farzaneh , Takai Hideki , Stein Gary , Galindo Mario , van Wijnen Andre J. 
Published: June 2021

Both genomic and non‐genomic mechanisms control the acquisition of specialized cellular phenotypes during development. When differentiated cells enter and exit mitosis, chromosomal condensation prevents gene expression. Post‐mitotic re‐activation of transcription is controlled in part by epigenetic marks on DNA and histones that control chromatin accessibility. However, mRNA molecules also appear to be transferred to progeny cells by either symmetrical or asymmetrical transmission. We have shown that the mRNA for the bone‐related transcription factor Runx2 mRNAs is elevated in the G2 phase of the cell cycle, but not translated in osteoblasts. This observation suggests that Runx2 protein is translationally rather than transcriptionally controlled after mitosis to maintain the phenotypic identity of osteoblasts. We used RNA‐seq expression profiling to assess whether there are other gene regulatory factors that are transmitted through mitosis. Our main results show that mRNAs for transcription factors are typically depleted during mitosis. For example, while Runx2 mRNA is retained in mitotic cells, expression of the closely related genes Runx1 and Runx3 is reduced. Strikingly, expression of Osterix/Sp7 and MEF2C, two transcription factors known to cooperate biologically with Runx2 in promoting the osteoblastic state, is selectively retained in mitotic cells. Our data suggest that selective somatic transmission of mRNAs for transcription factors in osteoblasts may be a general mechanism by which cells maintain their identity during cell division in skeletal development.Support or Funding InformationNIH R01 AR049069This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure and Mechanism of a Bacterial t6A Biosynthesis System
Authors: Luthra Amit , Swinehart William , Bayooz Susan , Phan Phuc , Stec Boguslaw , Iwata‐Reuyl Dirk , Swairjo Manal 
Published: June 2021

The universal N(6)‐threonylcarbamoyladenosine (t6A) modification at position 37 of ANN‐decoding tRNAs is central to translational fidelity. In bacteria, t6A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD, and TsaE. Despite intense research, the molecular mechanisms underlying t6A biosynthesis are poorly understood. Here we report biochemical and biophysical studies of the t6A biosythesis system from Thermotoga maritima. Small angle X‐ray scattering analysis reveals a symmetric 2:2 stoichiometric complex of TsaB and TsaD (TsaB2D2), as well as 2:2:2 complex (TsaB2D2E2), in which TsaB acts as a dimerization module, similar to the role of Pcc1 in the archaeal system. The TsaB2D2 complex is the minimal platform for the binding of one tRNA molecule, which can then accommodate a single TsaE subunit. Kinetic data demonstrate that TsaB2D2 alone, and a TsaB2D2E1 complex with TsaE mutants deficient in ATPase activity, can catalyze only a single cycle of t6A synthesis, providing evidence that TsaE‐catalyzed ATP hydrolysis is responsible for the turnover of product tRNA. Based on these results, we propose a model for t6A biosynthesis in bacteriaSupport or Funding InformationThis work was supported by the National Institutes of Health (GM11058)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the effect of distal charges on the reduction potential of the [2Fe‐2S] cluster in the Rieske protein from Thermus thermophilus
Authors: Munoz Janett , Hunsicker‐Wang Laura 
Published: June 2021

The Rieske Protein is characterized by a [2Fe‐2S] cluster that is ligated by two histidines (His154 and His134) and two cysteines (Cys151 and Cys132). The Rieske Protein is a subunit in complex III of the Electron Transport Chain (ETC) that helps shuttle electrons. Therefore, understanding its reduction potential is an important part of understanding the overall mechanism of the ETC. In the literature, reduction potential differences in Rieske have been proposed to be due to hydrogen bonding to the cluster and ligands as well as accessibility of the solvent and proximity of charged residues to the [2Fe‐2S] cluster. A new hypothesis poses that changes to charges on distal residues to the cluster will affect the reduction potential. To probe this hypothesis, a series of mutants have been produced in the Rieske protein from Thermus thermophilus. These mutants are grouped into three categories. First, mutants that remove positively charged residues (Arg or Lys) and replace them with neutral or negatively charged residues, have been produced. These changes result in a protein that is overall more negative. The outcome of these mutations are proteins with reduction potentials that are lower, and also point to an effect of distance from the cluster. The second and third groups of mutants remove either negatively charged amino acids (Glu and Asp) or neutral amino acids (Thr and Val) and replace them with either neutral or positive amino acids. These mutations cause the protein to be overall more positively charged, which should cause an increase in the reduction potential. All of these mutants are characterized by measuring the reduction potential, determining the pKa and pI values for the protein, and reacting the protein with diethyl pyrocarbonateThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Snapshots of C‐S cleavage in Egt2 reveals substrate specificity and reaction mechanism
Authors: Irani Seema , Naowarojna Nathchar , Liu Pinghua , Zhang Yan 
Published: June 2021

Ergothioneine is a thiol histidine derivative with a significant potential for biomedical applications. The sulfur incorporation strategy in ergothioneine biosynthesis differs from other well characterized transsulfuration reactions. A combination of a mononuclear non‐heme iron dependent enzyme‐catalyzed oxidative C‐S bond formation and a subsequent pyridoxal 5′‐phosphate (PLP)‐mediated C‐S lyase reaction leads to the net transfer of a sulfur atom from a cysteine to a histidine in the ergothioneine biosynthesis pathway. In this study, we have structurally and mechanistically characterized a PLP‐dependent C‐S lyase Egt2 (from Neurospora crassa), which mediates the sulfoxide C‐S bond cleavage in the ergothioneine biosynthesis pathway. The cation‐π interaction between substrate and enzyme accounts for Egt2's preference of sulfoxide over thioether as a substrate. Using mutagenesis and structural biology, we captured three distinct states of the Egt2 C‐S lyase reaction cycle, including a labile sulfenic acid intermediate captured in Egt2 crystals. Chemical trapping and high resolution mass spectrometry were used to confirm the involvement of the sulfenic acid intermediate in Egt2 catalysis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure and Function of Ergothionase, an Ergothioneine TMA‐Lyase from the Soil Bacteria Burkholderia sp. HME13
Authors: Medellin Brenda P. , Wang Shu , Liu Pinghua , Zhang Yan Jessie 
Published: June 2021

Since its discovery over a hundred years ago, L‐Ergothioneine (ET) has gained increasing importance as a powerful antioxidant shown to be beneficial for human health. Although this thione‐histidine derivative is only produced by a group of fungi and bacteria, humans have evolved a highly specific transporter (OCTN1) to absorb this molecule through diet and to accumulate it in certain tissues. In order to uncover the physiological role of Ergothioneine in human health, scientists in the field have attempted to study and understand the relationship between tissue‐specific ET accumulation and disease. However, multiple biological metabolites often interfere with the existing chemical detection assays, which cause skewed measurements and lead to inconsistencies in the ET levels reported. The need for a consistent, quick and highly selective method of ET detection is of upmost importance for the proper study of the role of ET in human physiology.The enzyme Ergothionase, found in the soil bacteria Burkholderia sp.HME13, performs the first degradation step for ergothioneine. Ergothionase carries out the cleavage of a C‐N bond, which produces thiourocanic acid and the organic cation trimethylamine (TMA), classifying it as a TMA‐Lyase. Thiourocanic acid has a strong absorbance at 311 nm, therefore it can be used to quantify the substrate concentration based on product formation. In this study we have characterized the structure and function of Ergothionase bound to its substrate, ET. The crystal structure for Ergothionase was solved using heavy metal derivatives and single anomalous diffraction. The ergothioneine bound structure reveals a tetrameric unit (biological unit) that shows structural similarities to the Histidine Ammonia Lyase/Phenylalanine Ammonia Lyase family (HAL/PAL). However, the characteristic MIO (3,5‐dihydro‐5‐methylidene‐4H‐imidazol‐4‐one) cofactor that carries the ammonia lyase activity in HAL/PAL enzymes is absent in ET TMA‐lyase. Therefore, a general acid base mechanism is proposed based on the substrate bound structure and active site environment. These structural and mechanistic studies can be a helpful guide in the development of a robust enzyme based assay for ET detection.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.