bluuebunny/crossref_metadata_2025 huggingface dataset:
Hippocampal CA1 Neuronal and Sympathetic Nerve Activities in Response to Restraint Water‐Immersion Stress in Conscious Rats
Authors: Nagao Kana , Ukita Hiroka , Yoshimoto Misa , Miki Kenju
Published: June 2021

The hippocampus has been suggested to play a significant role in adaptation to chronic stress. However, there has been lack of direct evidence for responses of hippocampal neuronal activity to chronic stress. The present study was aimed to record hippocampal CA1 neuronal and sympathetic nerve activities during chronic stress. Wistar rats were instrumented chronically with a combined probe made with multiple electrodes (100 micrometer stainless steel wires) for measurement of hippocampal CA1 neuronal activity and glass fiber for measurement of hippocampal cerebral blood flow (CBF), electrodes for measurements of renal (RSNA) and lumbar (LSNA) sympathetic nerve activity, and electroencephalogram, electromyogram, and electrocardiogram and with catheter for measurement of systemic arterial pressure (AP). Rats were exposed repeatedly to restraint water‐immersion stress (RWIS) for 90 min/day over 5 days. Hippocampal CA1 neuronal activity, RSNA, and LSNA were measured continuously and simultaneously before, during, and after RWIS. Hippocampal CA1 neuronal activity decreased immediately after onset of RWIS and the decreased level was maintained throughout the 90 min of RWIS on the first day of stress exposure. On day 5 of RWIS, hippocampal CA1 neuronal activity decreased immediately after onset of RWIS, however it gradually recovered to the control level during the RWIS period. Thus, hippocampal CA1 neurons gradually adapted to the repeated RWIS exposure while RSNA and LSNA increased immediately after the onset of RWIS and showed no adaptive response during repeated exposure to RWIS.

Support or Funding Information

JSPS Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MASP‐3: a new leptomeningeal protein in the lectin pathway
Authors: Dorta‐Contreras Alberto Juan , Padrón‐González Alexander Ariel , González‐Losada Cristobal , Lumpuy‐Castillo Jairo , Rodriguez‐Pérez Jose Alejandro , Ramos‐Robledo Alejandro , Martínez‐Reyes Josue , Schmitz Matthias , Zerr Inga , Hansen Anette Gudmann , Jensenius Jens Christian
Published: June 2021

Introduction

MASP‐3, a protein of the innate immune response is attracting increasing interest, in particularly in relation to its regulatory function in the lectin pathway.

Objective

Provide data that discriminate the source of MASP‐3 in cerebrospinal fluid.

Materials and methods

60 serum and cerebrospinal fluid samples from normal controls without organic disease with normal barrier function were obtained. MASP‐3 was assayed in samples of cerebrospinal fluid and serum with an ELISA, coated with anti MASP‐3 antibodies. It takes into account three variables: the molecular size‐dependent concentration gradient between CSF and blood, the variation in transfer between blood and CSF, and the CSF MASP‐3 concentration correlation with the albumin CSF/serum quotient (Qalb), i.e., with CSF flow rate.

Results

MASP‐3 concentration in CSF was lower than its serum concentration but MASP‐3 concentration in CSF was at least three‐fold higher than expected for a molecular‐size dependent passage from blood. The smaller inter‐individual variation of MASP‐3 concentrations in CSF of the control group (CV =0,396) compared to the MBL concentrations in serum (CV=0.413) indicate an independent source of MASP‐3 in CSF. The absolute MASP‐3 concentration in CSF increases with increasing Qalb. Among brain‐derived proteins in CSF only the leptomeningeal proteins showed a (linear) increase with decreasing CSF flow rate in comparison to neuronal and glial proteins that are invariant to changes of Qalb.

Conclusions

MASP‐3 in CSF is predominantly brain‐derived and all results pointed to the leptomeningeal cells as the source of the protein.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tissue Factor Enhances the Alveolar Epithelial Barrier Integrity during Acute Lung Injury
Authors: Sucharski Holly , Putz Nathan , Shaver Ciara , Ware Lorraine , Bastarache Julie
Published: June 2021

Introduction

We have previously reported that lung epithelial tissue factor (TF) knockout (TFΔEpi) mice have increased alveolar capillary barrier permeability. Ultrastructurally, these mouse lungs have profound morphological defects in epithelial attachment to the basement membrane. Adhesion/migration are regulated in part by integrins which bind the extracellular matrix proteins to modulate cytoskeletal dynamics. In cancer cells, TF binds β1 integrin and increases cell migration, a finding that suggests a role for non‐coagulative mechanisms of TF in cell adhesion and migration. We hypothesize that TF‐β1 integrin binding promotes alveolar epithelial barrier integrity through cell adhesion and migration during acute lung injury (ALI).

Method

TF overexpressing (TF+Epi) and knockout (TFΔEpi) mice, along with TF knockout A549 cells (A549ΔTF) were used to test whether lung epithelial TF is critical for the integrity of the alveolar epithelial barrier. TF+Epi mice were treated with intratracheal PBS or LPS and lung permeability was measured by bronchoalveolar lavage (BAL) fluid protein level. Alveolar type II epithelial cells (ATII) were isolated from TFΔEpi and TF+Epi mice to measure β1 integrin protein levels. A549ΔTF cells were treated with LPS (100ug/mL) or PBS for 24 hours before western blot of β1 integrin expression. Migration was assessed in wild‐type and A549ΔTF cells using Electric Cell‐substrate Impedance Sensing (ECIS) on cell monolayers. Adhesion was assayed by plating wild‐type and A549ΔTF cells on matrigel and quantifying the absorbance of crystal violet stained adherent cells.

Results

TF+Epi mice had decreased lung permeability (BAL protein 303 μg/mL +/− 62 versus 416 +/− 137 in control, p=0.025) following LPS challenge. A549ΔTF cells had significantly decreased cell adhesion (Figure) (absorbance 0.308 +/− 0.119 versus 0.603 +/− 0.246 in wild type, *p<0.001) along with decreased migration (p=0.017) compared to control A549 cells with wild type TF expression levels. A549ΔTF cells had decreased β1 integrin protein levels (relative expression 0.017 +/− 0.01 versus 0.61 +/− 0.02 control, p=0.036) following LPS challenge. ATII cells isolated from TFΔEpi mice also have decreased β1 integrin protein levels.

Conclusions

TF contributes to alveolar barrier integrity in part through a mechanism dependent on β1 integrin. These data provide support that lung epithelial TF may play a non‐coagulative role in protection during ALI. This novel role for TF in maintaining alveolar epithelial barrier integrity offers a potential therapeutic target during ALI.

Support or Funding Information

Vanderbilt University Initiative for Maximizing Student Diversity funding through the NIH, Nashville Veterans Affairs Medical Center, Funding: HL090785, HL103836

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tumor Necrosis Factor alpha (TNFα) reduces intestinal ascorbic acid (AA) uptake
Authors: Subramanian Veedamali S. , Sabui Subrata , Subramenium Ganapathy A. , Marchant Jonathan S. , Said Hamid M.
Published: June 2021

Intestinal inflammation and systemic bacterial infection are associated with an increase in the level of proinflammatory cytokines like tumor necrosis factor alpha (TNFα) in the intestinal mucosa and blood. Though important for intestinal immunity, TNFα also exerts profound effects on intestinal absorptive and secretory functions through specific intracellular signaling pathways like NF‐κB. Studies have shown that high levels of TNFα are linked to the pathogenesis of diarrhea in inflammatory bowel disease (IBD) and that these patients have low levels of vitamin C. To date, however, there has been little known about the effect of TNFα on intestinal absorption of vitamin C. Ouraim in this study was to address this issue using in vitro (Caco‐2), in vivo(mice), and ex vivo (enteroids) models, together with an array of physiological and molecular biological techniques. TreatingCaco‐2 cells and mice with TNFα led to a significant inhibition of 14C‐AAuptake compared to untreated controls. This inhibition was associated with significant decreases in SVCT‐1 protein, mRNA and hnRNA levels in TNFα treated Caco‐2 cells, mouse jejunum, and enteroids compared to their appropriate controls. TNFα also caused a significant inhibition in the activity of the SLC23A1 promoter in Caco‐2 cells. Further, treating Caco‐2 cells with the NF‐κB inhibitor celastrol blocked the inhibitory effect caused by TNFα on AA uptake, SVCT‐1 protein and mRNA expression, as well as SLC23A1 promoter activity. Finally, treatment of Caco‐2 cells with TNFα lead to a significant inhibition in the expression of the hepatocyte nuclear factor‐1α (HNF1α), which is known to drive the transcription of SLC23A1 promoter. Together, these findings demonstrate that TNFα inhibits intestinal AA uptake and that this effect is exerted, at least in part, at the level of transcription of the SLC23A1 gene through NF‐κB signaling pathway.

Support or Funding Information

Supported by NIH grants DK 107474, DK 58057, DK 56057 and a grant from the DVA.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
GluN2A Haploinsufficiency: a Novel Pre‐Clinical Model of Epilepsy
Authors: Wyllie David J. , Booker Sam A. , Yasmin Farhana , Marshall Grant , Marwick Katie F. , Wood Emma R. , Kind Peter C. , Hardingham Giles E.
Published: June 2021

N‐methyl‐D‐aspartate receptors (NMDARs) are ionotropic glutamate receptors that play pivotal physiological roles during development and maturation of the mammalian central nervous system. Their functional diversity is controlled by nature of the two GluN2 subunits that co‐assemble with two GluN1 subunits in the receptor‐complex. Four GluN2 subunits exist (GluN2A–D) and their expression patterns are developmentally and spatially restricted. In the mammalian forebrain the predominant NMDAR composition is one of a diheteromeric assembly of two GluN1 with (i) two GluN2A subunits (GluN1/2A) or (ii) two GluN2B subunits (GluN1/2B) or a triheteromeric assembly of two GluN1 and one of each of a GluN2A and GluN2B subunit. The GluN2 identity strongly influences NMDAR kinetic behaviour – GluN1/2A and GluN1/2A/2B NMDARs have faster deactivation rates than those exhibited by GluN1/2B. Thus loss or reduced expression of GluN2A will change the kinetic profile of NMDAR populations. It is now recognized that humans who have certain mutations in GluN2A NMDAR subunits or are haploinsufficient for GluN2A protein experience epileptic seizures. To investigate the mechanistic basis of neuronal dysfunction associated with GluN2A reduction we have created a novel transgenic rat line using CRISPR/Cas9 technology, where exon 8 of GluN2 is deleted. We have studied the intrinsic and synaptic properties of hippocampal CA1 pyramidal neurons obtained from rats either completely lacking expression of Glun2A (Grin2A−/−) or ones which are heterozygous for the deletion (Grin2A+/−). Whole‐cell patch‐clamp recordings revealed that compared to recordings from wild‐type (WT) neurons no significant differences are observed in intrinsic excitability of either Grin2A+/− or Grin2A−/− pyramidal neurones when recordings were made from postnatal day 12–14 or postnatal day 28–32 animals. In addition, miniature and spontaneous excitatory and inhibitory synaptic currents recorded from either WT, Grin2A+/− or Grin2A−/− neurons showed little differences in the amplitudes or frequency of events. As is to be expected, however, loss of GluN2A expression leads to a slowing of the duration of the NMDAR‐mediated component of evoked synaptic currents. Our data also indicate that the magnitude of synaptic plasticity at Schaffer collateral/commissural synapses on to CA1 pyramidal neurons is reduced.

Support or Funding Information

Biotechnology and Biological Sciences Research Council BB/N015878/1 Epilepsy Research UK P1602

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Urocortins on Intestinal Ion Secretion in the Mouse Colon
Authors: Liu Sumei , Karo Aaron
Published: June 2021

Background and Aims

Urocortins (Ucn1, Ucn2, and Ucn3) belong to the corticotropin‐releasing factor (CRF)‐like peptide family and bind to two subtypes of CRF receptors (CRF1 and CRF2) to exert their biological effects. Ucn1 binds to both CRF1 and CRF2 receptors with equal high affinity, whereas Ucn2 and Ucn3 bind exclusively to CRF2. CRF and urocortins are expressed in the gastrointestinal tract. CRF has been implicated in stress‐stimulated intestinal ion secretion and diarrhea. However, the effects of urocortins on intestinal ion secretion have remain uninvestigated. The aim of the present study was to investigate the role of urocortins in the regulation of intestinal ion secretion.

Methods

Mucosa/submucosa preparations from mouse colon were mounted in Ussing flux chambers for measurement of short‐circuit current (Isc) as an indicator of mucosal ion secretion.

Results

Application of CRF, Ucn1, Ucn2, and Ucn3 to the basolateral side of the preparation increased baseline Isc in a concentration‐dependent manner with an EC50 (in M) of 1.28×10−6, 6.04×10−7, 2.89×10−7, or 2.98×10−7, respectively, with the rank order of potency: Ucn2≈Ucn3>Ucn1>CRF. At 1 μM, Ucn3 caused a greater rise in Isc (ΔIsc: 47.01±8.94 μA/cm2), compared with Ucn2 (17.71±2.63 μA/cm2), Ucn1 (7.06±4.33 μA/cm2), or CRF (8.92±4.11 μA/cm2). The CRF1 receptor antagonist NBI 27914 significantly suppressed Ucn1‐evoked ΔIsc, but had no effect on Ucn2‐ or Ucn3‐evoked ΔIsc. The CRF2 receptor antagonist antisauvagine‐30 significantly suppressed Ucn1‐evoked ΔIsc, and completely blocked Ucn2‐ or Ucn3‐evoked ΔIsc. The neuronal blocker tetrodotoxin had no effect on Ucn1‐, Ucn2‐, or Ucn3‐evoked DIsc.

Conclusions

The results suggest that urocortins stimulate colonic ion secretion independent the submucosal plexus of the enteric nervous system. Both CRF1 and CRF2 receptors are involved in Ucn1‐stimulated ion secretion, while Ucn2‐ and Unc3‐stimulated ion secretion is mediated by the CRF2 receptors. Ucn2 and Ucn3 are more potent than CRF in stimulation of colonic ion secretion and may play a dominant role in stress‐induced hyper secretion and diarrhea.

Support or Funding Information

NIH R15 DK097460‐01A1 (SL) and UWL graduate research service and educational leadership grant (AK).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Knockout of Txnip in the Intestinal Epithelial Cells Abrogates the High Fat Diet‐Induced Fructose Uptake in Mice
Authors: Shah Anu , Dagdeviren Sezin , Hundal Henna , Lee Richard T.
Published: June 2021

Thioredoxin interacting protein (Txnip) was initially identified as a protein that can bind to and inhibit thioredoxin, but it is now recognized to participate in nutrient sensing and carbohydrate metabolism. We recently discovered that Txnip binds to the fructose transporters, Glut 2 and Glut 5, to increase the fructose uptake by enterocytes. Additionally, we demonstrated that mice with diabetes induced by streptozotocin exhibit significantly higher fructose absorption compared to their non‐diabetic counterparts. Interestingly, this increase of fructose absorption in the diabetic state requires Txnip. In the current study, we studied the effects of a high fat diet (HFD) on fructose uptake and the involvement of Txnip in this process while the mice were still euglycemic. We hypothesized that high fat diet (HFD) feeding would increase fructose absorption by the jejunum compared to the controls on standard chow diet (RD), and this increase would be prevented in mice with deletion of Txnip in the intestinal epithelium despite being on a HFD. To test this hypothesis, 8‐week‐old male C57BL/6J wild‐type mice were kept on HFD for 16 weeks. At the end of 16 weeks, HFD‐fed mice not only developed insulin resistance as expected, but also displayed significantly higher fructose absorption (hepatic portal vein blood: p<0.01 and liver: p<0.05 vs regular diet mice, n=6–7 mice/group) 20 minutes after the oral gavage of [14C‐(U)]‐D‐fructose. Interestingly, gene and protein expressions of Txnip were also significantly higher in the jejunal samples from these mice, suggesting a potential role of Txnip in fructose uptake by the HFD‐fed mice. Since fructose absorption primarily occurs in the jejunum, using a cre‐lox breeding strategy, we generated a Txnip villin‐cre mice that lacks Txnip in the intestinal epithelial cells; and then subjected these mice and their cre counterparts to RD/HFD at 7–9 weeks of age. The preliminary data at 1 month post‐diet shows a downward trend of fructose absorption in both RD‐fed and HFD‐fed Txnip villin cre mice compared to cre‐controls that exhibited a physiological level of Txnip in villi. Therefore, our data indicate that HFD facilitates fructose uptake by the intestine, and targeted deletion of Txnip at the intestinal level can decrease absorption of dietary fructose. This finding suggests a molecular mechanism by which a high fat diet can promote additional hepatic lipid synthesis through increased fructose absorption.

Support or Funding Information

National Institute of Health (RTL R01DK107396) and Canadian Diabetes Association (PF‐3‐16‐5176‐AS to AS)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
c‐Src mediates intestinal stem cell response to high fat diet
Authors: Theiss Arianne L. , Han Jie , Jackson Dakota L. , Park Junhyoung , Jung Kwang Hwa , Kaipparettu Benny A.
Published: June 2021

c‐Src is a commonly upregulated oncogene in colorectal cancer. High fat diet (HFD) augments the numbers and function of Lgr5+ intestinal stem cells (ISCs), increasing the capacity of these stems cells to initiate tumors. Discoveries in triple negative breast cancer models and patient specimens suggest that mitochondrial reprogramming to fatty acid oxidation (FAO) activates c‐Src. Here, we determined the role of FAO induced by HFD on c‐Src activation and ISC self‐renewal. Lgr5‐EGFP‐IRES‐creERT2 (Lrg5‐EGFP) knock‐in mice were fed a dietary chow consisting of 45% kcal fat or a low fat matching diet ad libitum from 8 to 20 weeks of age. Beginning at age 10 weeks, mice were i.p. injected with 20 mg/kg Etomoxir (ETX), an inhibitor of FAO, twice per week. Intestinal enteroids derived from Lgr5‐EGFP mice were treated with constituents of HFD (30 μM palmitic acid and 30 μM oleic acid) every 3 days for 2 weeks ± 20 μM PP2, a Src inhibitor. Numbers of intestinal Lgr5‐GFP+ cells were quantitated and isolated by flow cytometry and expression of c‐Src target gene Cyclin D1 and ISC self‐renewal gene Olfm4 were measured by qPCR. c‐Src activation was measured in human colon cancer cell lines HCT‐116 and Caco2 treated with 200 μM ETX or 10 mM L‐carnitine (FAO activator) for 16 hr. HFD increased the number of Lgr5+ ISCs and expression of Cyclin D1 and Olfm4, which were prevented by ETX. PP2 inhibition of c‐Src in enteroids prevented the palmitic acid/oleic acid‐induced increase in Lgr5+ ISC numbers. ETX decreased and L‐carnitine induced c‐Src activity in HCT‐116 and Caco2 cells. Our data provide novel evidence that the Src pathway plays an important role in ISC response to constituents of HFD.

Support or Funding Information

Collaborative Faculty Research Investment Program (CFRIP), Baylor Scott & White Health and Baylor College 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.
Dietary Fat Quality Plays a Sex‐Specific Role in Attenuating Metabolic Risk Factors in Aging CD‐1 Mice
Authors: Unger Allison , Jetton Thomas , Kraft Jana
Published: June 2021

Diet quality (e.g., dietary fat type) and aging play an important role in the development of type 2 diabetes, however, is not known if long‐term consumption of diets with differing fatty acid profiles can modulate metabolic risk factors in an aging population. Accordingly, we aimed to examine how different sources of fatty acids added to a western‐style diet might improve metabolic risk factors during aging. Sixty male and female outbred CD‐1 mice (n = 10/group) were fed isoenergetic high‐fat diets (40% kcal fat) containing either (1) American diet fat blend (100%) (Control, CON; reflecting the average fatty acid profile consumed in the United States linked to dysmetabolism and increased inflammation), (2) CON fat (70%) + 30% butter oil (BO), (3) CON fat (70%) + 30% echium oil (EO), or (4) CON fat (70%) + 30% fish oil (FO) for a total of 14 months. Standard physiological measurements and glucose homeostatic assays (i.e., intraperitoneal insulin and glucose tolerance tests) were performed at regular intervals (3, 6, 9, and 12 months of dietary intervention) and key tissues were harvested at 14 months of age. Despite CON males having a consistently lower feed intake (P < 0.001) and weight gain (P = 0.002), they exhibited a markedly reduced glucose tolerance by 9 months (P = 0.02) compared to FO (P = 0.005) and EO males (P < 0.001) which persisted through 12 months. CON males also had an increased accumulation of triacylglycerols in the liver compared to EO, BO, and FO males at 14 months (P < 0.001). Although BO males showed transiently reduced glucose tolerance at 9 months (P = 0.02) compared to FO (P = 0.009) and EO males (P = 0.001), this phenotype dissipated by 12 months. EO males exhibited a trend of overall improved insulin sensitivity compared to BO males (P = 0.05). While females displayed no differences in feed intake, weight gain, glucose tolerance, or insulin sensitivity among the diet groups at 3, 6, and 9 months, unexpectedly, by 12 months, all groups displayed improved glucose tolerance and insulin sensitivity compared to earlier time points (P < 0.001 and P = 0.02, respectively). Our study provides evidence that dietary fat quality impacts metabolic risk factors in a sex‐specific manner.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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 Information

This 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 Information

American 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 Information

NIH/NIDCR R01DE022804

This 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 Information

This 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 Information

NIH R01 AR049069

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 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 bacteria

Support or Funding Information

This 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 pyrocarbonate

This 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.
Engineering of Enzymes to Improve Lignin Breakdown for Use in Fuel Ethanol Production
Authors: Greene Russell D. , Hall Bonnie L.
Published: June 2021

Lignin is the second most abundant biopolymer on Earth, behind cellulose. Lignin contains aromatic rings similar to those in fossil fuels, and breaking down lignin into smaller polymers is the first step in using it as a source of fuel. Once lignin is broken down, additional enzymes can then hydrolyze the cellulose and hemicelluloses, so that sugar fermentation and production of ethanol can occur. Lignin is difficult to break down, although some fungi can slowly degrade it using lignin peroxidase and/or laccase enzymes. Laccases break down the complex structure of lignin, while lignin peroxidases oxidize the aromatic rings. These are slow processes and are not practical for mass‐producing fuels from lignin. We are engineering these enzymes with the goal of improving the efficiency of lignin degradation. We have begun by studying BglB, an enzyme involved in the hydrolysis of cellulose, as a proof of concept. We have successfully expressed, purified, and assayed the activity of the BglB protein. We have also used FoldIt software to identify mutations for modifying the activity of the BglB protein. One specific series of mutants characterized had the amino acid substitutions W402S, W412F, E409Q, and W410H. We have characterized the activity of these mutants, and found W412F had similar activity as the wild type, while the activities of W402S, E409Q, and W410H were lower than wild type. We are now ready to expand these engineering methods to lignin peroxidase and laccase enzymes, to generate more efficient enzymes for the production of fuel ethanol from lignin.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.