bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein methylation and translation: Role of lysine modification on the function of yeast elongation factor 1 alpha
Authors: White Jonelle , Deramchi Neil , Cato Tieranee , Gabunilas Jason , Chanfreau Guillaume , Clarke Steven
Published: June 2021

Proteins of the translational apparatus, including ribosomal proteins, release factors, and elongation factors, are known to be modified by a large number of methyltransferases in eukaryotic cells. The majority of these methylation reactions occurs at lysine residues. To date, 12 protein lysine methyltransferase involved in these reactions (Efm1–7; Rkm 1–5) have been identified in the yeast Saccharomyces cerevisiae. Of these 12, 5 appear to be specific to elongation factor 1 alpha (EF1A)(Efm1, Efm4–7). Each methyltransferase modifies a specific lysine residue to give mono‐, di‐, and tri‐methyllysine products. Two of these enzymes (Efm4 and Efm5) are highly conserved in nature while the others appear to be fungal specific.

In S. cerevisiae the functional implications of lysine methylation are mostly unknown. EF1A's primary function, with its GTP cofactor, is to deliver amino‐acyl tRNA to the A site of the ribosome during translation. Once the correct tRNA is positioned, a conformational change results in the hydrolysis of GTP and its release from the ribosome for another cycle. Studies have also shown the involvement of EF1A in the assembly of ribosomes.

Using yeast genetics we have assessed the biological relevance of EF1A lysine methylation on its translational roles using two approaches. The first approach uses site directed mutagenesis to mutate four of the most conserved methylated lysine sites to arginine residues. The second approach involves combinatorial knockouts of the genes that code for each of the five methyltransferases. In each approach, we have then used functional translation assays in yeast to evaluate the physiological impact of the loss of EF1A methylations. We have found that inactivation of multiple methyltransferases can cause a significant reduction of translational fidelity. Additionally, we have shown that the ribosomal assembly function of EF1A does not appear to be affected by the loss of methyltransferase activity. These studies should illuminate why one protein appears to need five different methyltransferases for its functions.

Support or Funding Information

This work is funded by the National Science Foundation Grant MCB‐1714569 and NSF‐LSAMP Bridge to Doctorate Fellowship.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Studies of the Iterative Enzyme DesD: Cooperativity, Substrate Variability and Conformational Changes
Authors: Mojab Yasi , Hoffmann Katherine
Published: June 2021

Iron is essential for bacteria to survive. When iron concentration is low, bacteria secrete siderophores. Siderophores can be assembled via two pathways. They may be assembled by non‐ribosomal peptide synthetase (NRPS)‐ dependent pathway, which uses large complexes and polyketide pathways, or NRPS‐independent siderophores known as NIS‐pathway, which uses small enzymes to make peptide bonds between cellular building blocks. In our lab, we focus on non‐ribosomal peptide synthase independent siderphore (NIS), because they are associated with virulence in pathogenic bacteria. Streptomyces coelicolor produces NIS siderophores Desferrioxamines B and Desferrioxamines E via the same pathway containing four enzymes: DesA, DesB, DesC, and DesD. In this pathway, DesD is a NIS synthetase, a model enzyme for a novel family utilizing unique structure and chemistry. Atomic‐level structures of DesD in both apo and substrate‐bound form show a flexible loop capping the binding site, half‐site occupancy, and a series of conserved catalytic residues in contact with the substrate. Analysis of these may reveal the source of cooperativity, variations in substrate association, and mechanism. Future work will include structure‐based inhibitor design to prevent DesD from binding to substrate and thus prevent synthesis of a virulent factor of pathogenic bacteria.

Support or Funding Information

This work was supported by a grant from the National Science Foundation (MCB ‐ 1716986) to K.M.H, and by California Lutheran University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transport and synthesis of a bacterial natural product
Authors: Dassama Laura M. K. , Kenney Grace E. , Rosenzweig Amy C.
Published: June 2021

Nature is replete with natural products that are pharmacological agents, secondary metabolites, and chemical agents, among others. A common class of natural products is those that bind metals and are taken into cells to fulfill the cell's metal needs. A large majority of these metallo‐natural products bind iron and are referred to as siderophores (iron “carriers”). Similar molecules that bind copper rather than iron have been discovered recently. These molecules were initially isolated from the growth media of methanotrophic bacteria (bacteria that metabolize methane), which have an elevated copper requirement. These “chalkophores” (copper “carriers”) have been termed methanobactins (Mbns). Mbns are secreted when methanotrophic bacteria are cultured under conditions of limiting copper. Mbns acquire copper from the environment and are taken up into the cell. Mbns have a high affinity for copper, and this has sparked interest in their potential use for the treatment of diseases of toxic copper overload. Since its discovery over a decade ago, the mechanisms of Mbn synthesis, transport and its contribution(s) to the overall metal homeostasis of methanotrophic bacteria have been the focus of intensive investigations. This work describes the enzyme machinery responsible for its transport and biosynthesis.

Support or Funding Information

NIH grants GM118035 (A.C.R) and F32GM110934 (L.M.K.D.), NSF grant MCB0842366 (A.C.R.), AHA 14PRE20460104 (G.E.K), and a Burroughs Wellcome Fund PDEP award (L.M.K.D.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altering the Specificity Properties of 2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase from N. asteroides A3H1
Authors: Smith Emily Elizabeth , Hoang Dylan , Cromwell Daniel , Watkins Linette
Published: June 2021

Biodesulfurization is a metabolically important process that occurs naturally in bacterial cells found near oil and coal deposits. In sulfur‐specific biodesulfurization, the dsz pathway, enzymes selectively remove the organic sulfur found in crude oil without disrupting the carbon backbone. The rate‐limiting step within this metabolic pathway is the conversion of 2‐(2′‐hydroxyphenyl)benzenesulfinate (HPBS) to 2‐hydroxybiphenyl using HPBS desulfinase (DszB). The DszB enzyme from Nocardia asteroides A3H1 was overexpressed in E. coli, purified and characterized kinetically. Homology studies identified an active site loop with conserved amino acids that could play a role in the specificity for different organosulfur compounds found in crude oil. Point mutations were generated to study the importance of amino acids 195 and 200 on the specificity of the enzyme. Three mutant enzymes (A195R, A200R and A195/200R) prepared using codon‐optimized gene constructs were co‐expressed with GroESL and purified from E. coli using Ni2+‐affinity chromatography. Substrate specificity experiments were performed to define the role of the active site loop on specificity. Kinetic measurements indicate a decreased specificity for HPBS. Fixed‐time sulfite assays indicate an increased preference for smaller ringed substrates, demonstrating the importance of amino acids 195 and 200 and defining the specificity of the enzyme.

Support or Funding Information

Supported by NSF grant, CHE 1461175, and James Madison University Department of Chemistry and Biochemistry

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
1. A light inducible gene activation system toward controllable cell‐based therapeutics
Authors: Huang Ziliang , Wu Yiqian , Pan Yijia , Allen Molly , Chang Ya‐Ju , Chien Shu , Wang Yingxiao
Published: June 2021

T cells engineered to express chimeric antigen receptors (CARs) on cell surface can recognize and engage with target cancer cells with redirected specificity. This CAR T cell‐mediated immunotherapy is under intensive investigation as a revolutionary therapy for cancer. However, adverse effects have been reported, including on‐target off‐tumor cytotoxicity, cytokine release syndrome and tumor lysis syndrome. One of the solutions is to engineer on/off‐switchable cells to achieve high‐precision control over their activation. Optogenetics utilizing genetically encoded dimerizers has been applied to remotely activate gene expression. Here, we developed a new blue light‐mediated light‐inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control cell activation, by integrating a LOV2‐based light‐inducible nuclear localization signal with CRY2‐CIB1 dimerization. We demonstrated light‐controllable gene expression and functional modulation in HEK 293T and Jurkat T cell lines. We further applied the LINTAD system in primary human CAR T cells and showed that the light‐stimulated cells possessed a significantly stronger cytotoxicity against target cancer cells than the non‐stimulated ones. We also extended the LINTAD system to activate the expression of the TNF‐related apoptosis‐inducing ligand (TRAIL) in HEK 293T cells, and observed a gain of cytotoxicity upon light stimulation. Therefore, our newly developed LINTAD system can serve as an efficient and general tool to remotely and non‐invasively control gene activation in live cells for therapeutic applications.

Support or Funding Information

This work is supported by grants from NIH HL121365, GM125379 (S.C., Y. Wang), CA204704, CA209629, NSF CBET1360341, DMS1361421 (Y. Wang), and UC San Diego.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biosynthesis of acyl‐CoAs sustains prostate cancer progression
Authors: Cai Houjian , Ma Yongjie
Published: June 2021

Objective of the study

Fatty acid metabolism is essential for the biogenesis of cellular components and ATP production to sustain proliferation of cancer cells. Bioconversion of exogenous or de novo synthesized fatty acids to their corresponding fatty acyl‐CoAs, which is required for the fatty acids to participate in metabolic processes, is catalyzed by long chain fatty acyl‐CoA synthetases (ACSLs). However, the influence of individual ACSL isoforms on the progression of prostate cancer remains unknown. The goal of this study is 1) to analyze how ACSLs regulates the biosynthesis of acyl‐CoAs, thereby prostate cancer progression.

Statement of Methods

Lentivirus carrying shRNA of ACSL1 was constructed and produced in house. Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) were measured by Seahorse. The amount of protein was evaluated by immunoblotting. The amount of acyl‐CoAs was analyzed by LC‐MS/MS. The amount of triglyceride and neutral lipid were measured by gas Chromatography and Oil red O staining, respectively. TCGA database was used for analysis of ACSL1 expression levels in different stages of prostate cancer. Additionally, inhibition of cell proliferation, migration, invasion assays and cell cycle analysis were analyzed in numerous cancer cells. Prostate xenograft tumor models were applied for in vivo studies. Prostate tumor tissue array was utilized for the analysis of ACSL1 expression.

Summary of the results

In this study, the correlation of ACSL expression levels and the amount of intracellular acyl‐CoAs was systematically analyzed. We demonstrate that expression levels of ACSL1 were significantly associated with the levels of a variety of acyl‐CoAs, and were elevated in human prostate tumors. ACSL1 increased the biosynthesis of fatty acyl‐CoAs including C16:0−, C18:0−, C18:1− and C18:2‐CoA, triglycerides and lipid accumulation in cancer cells. In addition, ACSL1 modulated mitochondrial respiration through regulation of carnitine palmitoyltransferase 1A (CPT1A) activity. Knockdown of ACSL1 expression inhibited the cell cycle, and suppressed the proliferation and migration of prostate cancer cells in vitro, and growth of prostate xenograft tumors in vivo.

Conclusion

Our study demonstrates ACSL1 as playing a role in prostate tumor progression. Our results provide a therapeutic strategy of targeting fatty acid metabolism for the treatment of prostate cancer.

Support or Funding Information

NIH (R01CA172495) and DOD (W81XWH‐15‐1‐0507)
Characterizing the physiologic effects of exogenous ketone supplements – an alternative or adjuvant to the ketogenic diet
Authors: Poff Angela , Koutnik Andrew , Deblasi Janine , Rogers Christopher , Kesl Shannon , Ward Nathan , D'Agostino Dominic
Published: June 2021

The ketogenic diet (KD) is a high fat, adequate protein, low carbohydrate diet that is used clinically for refractory pediatric epilepsy, and is under investigation as a treatment for a wide variety of disorders, including many neurological diseases, cancer, obesity, and diabetes. Studies suggest that many of the beneficial effects associated with the KD are mechanistically attributable to the ketone bodies, leading our lab and others to develop and test exogenous ketone supplements (KS). KS elevate blood ketones without the need for dietary restriction of carbohydrate. In order to further our understanding of this new technology, we sought to characterize the metabolic effects, safety, and toxicity of KS in healthy rats. KS tested include the R,S‐1,3‐butanediol acetoacetate diester, 1,3‐butanediol, medium chain triglyceride oil, caprylic acid, and beta‐hydroxybutyrate mineral salts, alone and in various combinations. Healthy rats were administered KS by oral gavage or in their food in an acute (once, or 7 days), sub‐chronic (1 month) or chronic (4 months) fashion and analyzed for a variety of metabolic and physiologic biomarkers. KS rapidly elevate blood ketones to therapeutic levels (2–5mM), and simultaneously decrease blood glucose. Some types of KS induce a mild calorie restriction effect, and preliminary evidence suggests ketone ester treatment during CR may preserve lean muscle mass in this catabolic state via a non‐insulin dependent mechanism. Satiety may be increased by KS as demonstrated by an elevation in leptin following KE administration, even when animals were self calorie‐restricting. KS significantly alters the global metabolome, indicating a shift from carbohydrate‐based to fat‐based metabolism. Some markers of this were an elevation in medium chain fatty acids, an increase in acetyl coA and acetylcarnitine suggestive of enhanced mitochondrial LCFA oxidation, and an increase in Kreb's cycle intermediates such as citrate, succinate, and fumarate. Inflammatory profiling following chronic, but not acute, KS administration revealed decreases in many pro‐inflammatory cytokines, including IL‐1β, IL‐6, IFN‐γ, MCP‐1, and RANTES. KS appears safe as there were no signs of toxicity or adverse changes in total cholesterol, HDL, LDL, triglycerides, or markers of liver/kidney function over the chronic treatment protocol. KS may be a useful alternative or adjuvant to the KD for disorders where nutritional ketosis is therapeutic.

Support or Funding Information

Office of Naval Research, Scivation Inc, Disruptive Nutrition, Epigenix Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Parathyroid Hormone Shows Novel Calcium Sensing Ability in Binding to Parathyroid Hormone 1 Receptor
Authors: Culhane Kelly J. , Yan Elsa C.Y.
Published: June 2021

Family B G protein coupled receptors (GPCRs) are important drug targets for metabolic diseases, such as diabetes and osteoporosis. Despite this importance, the structure and function of family B GPCRs remain poorly understood. We have developed a nanodisc purification protocol to purify family B GPCRs for molecular studies. Here, we focus on parathyroid hormone 1 receptor (PTH1R). PTH1R regulates homeostasis of Ca2+ and phosphate and is a drug target for osteoporosis. It has two native ligands, parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Truncated versions of both ligands, PTH(1–34) and PTHrP(1–36), can fully activate the receptor and PTH(1–34) is clinically used for osteoporosis treatments. In this study, we purified PTH1R in nanodiscs and used fluorescence anisotropy to examine ligand binding. We found that the affinity increases more than 5‐fold in the presence of 15 mM Ca2+ for PTH(1–34) but not PTHrP(1–36). This result carries physiological significance because PTH(1–34) but not PTHrP(1–36) is involved in bone remodeling process, in which Ca2+ concentrations can fluctuate up to 40 mM. We hypothesize that the Ca2+‐dependent binding is mediated by non‐conserved, negatively charged residues of both PTH(1–34) and PTH1R. In PTH(1–34), mutations of Glu19 and Glu22 that are not conserved in PTHrP(1–36) largely abolished the observed Ca2+ effect. In the receptor, mutations in residues Glu258, Glu259 and Glu260 in the extracellular loop 1 also largely abolish the Ca2+ effect. The results not only support the hypothesis but also demonstrate the unique ability of PTH(1–34) in sensing Ca2+ while activating PTH1R. This dual function of a peptide hormone is a novel observation in endocrine signaling. Understanding it can potentially reveal the complex role of PTH signaling in bone remodeling and improve the PTH(1–34) treatment for osteoporosis. Further investigations of the differences between PTH(1–34) and PTHrP(1–36) in response to Ca2+ during PTH1R activation will provide insights into molecular mechanism of ligand‐biased signaling process and allosteric interactions of family B GPCRs.

Support or Funding Information

This work was supported by an NSF career award to ECYY (MCB‐0955407) and KJC is supported by T32 GM008283‐27

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcium Dependent Properties of Ig 80–83 of N2A Titin
Authors: Thompson Blair Marie , Dutta Samrat , Monroy Jenna , Nishikawa Kiisa
Published: June 2021

Titin is a giant elastic protein responsible for passive force generation in muscles. In addition, numerous studies have implicated a role for titin in active muscle. Recent work on muscles from mdm mice, suggests that during muscle activation, the N2A region of titin binds to actin thereby increasing titin and active muscle stiffness. Atomic force microscopy (AFM) can be used to measure the elastic properties of a titin construct, specifically by unfolding and refolding of its constituent immunoglobulin (Ig) domains. We hypothesized that the Ig domains from the N2A region of titin (Ig 80–83) exhibit different elastic properties in the presence and absence of calcium. To test this hypothesis, we stretched a N2A‐like construct consisting of Ig 80–83 and a Halo‐tag domain at different pulling velocities, in the presence and absence of calcium. Our results suggest a non‐linear unfolding energy landscape for serially linked Ig 80–83 domains in the presence of calcium. We also found, while calcium increased the persistence length of unfolded Ig domains in the N2A‐like construct, its contour length did not change. Increase in persistence length indicates an increase in the stiffness of the N2A domain, which supports the hypothesis that titin stiffness increases in the presence of calcium. These results differ from previous studies without calcium and indicate unique properties of Ig domains specific to the N2A region of titin.

Support or Funding Information

APS IOSP Fellowship Awarded to B. Thompson, NSF IOS‐1451650 Awarded to J. Monroy

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insulin/IGF‐1 Enhances Intestinal Epithelial Crypt Proliferation through PI3K/Akt, and Not ERK Signaling in Obese Humans
Authors: Zhou Weinan , Rowitz Blair M. , Dailey Megan J.
Published: June 2021

The intestinal epithelium plays important roles in nutrient absorption, satiety hormone release and immune barrier function. In order to ensure proper tissue actions are maintained, the cells are continuously regenerated through proliferation and differentiation of stem cells located in the intestinal crypts. Obesity affects this process and results in greater stem cell proliferation and abnormal tissue growth and function. Obesity‐induced high levels of insulin/insulin‐like growth factor‐1 (IGF‐1) in the stem cell niche are found to impact the changes in proliferation in rodents indicating that insulin/IGF‐1 receptors may play a role in modulating intestinal epithelial stem cell proliferation. Insulin receptor resistance is a hallmark of obesity that can be driven by an imbalance between or abnormal signaling within two downstream signaling pathways, PI3K and ERK, in non‐intestinal epithelial tissues. To determine whether insulin/IGF‐1 can induce proliferation in human intestinal epithelial stem cells and if PI3K or ERK pathways are involved, we used primary small intestinal epithelial crypts and investigated 1) the effect of insulin/IGF‐1 on crypt proliferation, and 2) the effect of insulin/IGF‐1 signaling inhibitors (i.e. PI3K/Akt pathway inhibitor Wortmannin and ERK pathway inhibitor PD98059) on insulin/IGF‐1‐induced crypt proliferation. We found that insulin/IGF‐1 enhanced crypt proliferation in obese humans. Inhibition of PI3K/Akt pathway attenuated insulin/IGF‐1‐induced crypt proliferation, but inhibition of ERK pathway had no effect. These results suggest that the classical metabolic PI3K pathway and not the canonical proliferation ERK pathway is involved in insulin/IGF‐1‐induced increase in crypt proliferation in obese humans. Pathway‐specific impairments in insulin/IGF‐1 signaling may contribute to reciprocal relationships between insulin/IGF‐1 resistance and intestinal epithelial stem cell proliferation and differentiation that fosters tissue dysfunction.

Support or Funding Information

This work was supported by USDA Hatch (to MJD) ILLU‐538‐926.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Sphingosine‐1‐Phosphate Activity in Liver Preservation Injury
Authors: Fyffe‐Freil Ria Christian , Limkemann Ashley , Lindell Susanne L. , Mangino Mangino J.
Published: June 2021

Lipid signaling molecules, such as lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P), act as growth and survival factors, and may be involved in liver preservation injury. Recent studies have proposed that there is a cytoskeletal component of preservation injury involving cytoskeletal linker proteins called ERM proteins. In preservation injury, moesin (the predominant ERM protein in the liver) is dysregulated, and found in its inactive, closed conformation. We hypothesize that serum‐derived growth factors (like LPA and/or S1P) may control known upstream regulators of moesin, RhoA and Rho kinase (ROK), which ultimately control moesin functionality and cell adhesion to the basement membrane. To begin testing this hypothesis, we demonstrated that inhibition of RhoA and ROK potentiated preservation injury in hepatocyte models of hypothermic ischemia. Preliminary data suggests that adding exogenous LPA to hepatocyte culture conditions prior to hypothermic ischemia induction can rescue cell viability from preservation injury. We also observed cytotoxicity with low concentrations (0.1%) of bovine serum albumin (BSA), which is known to avidly bind and hold these lipid growth factors. Inhibition of S1P synthesis via selective pharmacological inhibition of sphingosine kinase 2 (SphK2) produced striking results (figure 1). In vitro, cell viability was significantly decreased upon SphK2 inhibition, and in vivo, rodents receiving a liver transplant after that liver was flushed with a solution containing a SphK2 inhibitor had 100% mortality, relative to only 20% control mortality. These data support the hypothesis that serum‐derived lipid signaling molecules, such as LPA and S1P, are protective during hypothermic ischemia, and removing these at the time of organ preservation and flushing may be harmful.

Support or Funding Information

NIH R01 DK087737

DoD W81XWH‐16‐2‐0040

DoD W80XWH‐17‐1‐0602

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The mammalian blastema and induced regeneration in mice.
Authors: Muneoka Ken
Published: June 2021

Fingertips of humans and toe tips of mice possess the capacity to regenerate following amputation injury. The mouse toe tip has been developed as a regeneration competent model to investigate the regeneration process, and digit amputation at a more proximal level serves as a non‐regenerative wound model to test strategies for enhanced regeneration. The process of toe tip regeneration is divided into a series of overlapping events beginning with an inflammatory response, a bone degeneration response, epidermal closure, migration and proliferation of progenitor stem cells, blastema formation and redifferentiation. These stages are distinct from wound healing of a non‐regenerative amputation that results in truncated skeletal elements covered with scar tissue. BMP signaling has been shown to be essential for toe tip regeneration and BMP treatment is effective in enhancing the regenerative capacity of proximal level amputations. The ability to induce a regeneration response from non‐regenerative amputation wound identifies an unrealized regenerative potential of cells present at traumatic wounds in mammals. To date, we have determined that the regeneration of bone, cartilage, synovial cavity, tendon and ligament can be induced from a non‐regenerative amputation wound with different regimens of targeted growth factor treatments. These studies provide a road map for developing strategies to control regeneration events in mammals and serve as a new direction for the field of regenerative medicine.

Support or Funding Information

DARPA (W911NF‐09‐1‐0305)

US Army Research Laboratory (W911NF‐09‐1‐0305)

Texas A&M University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Disturbed flow disrupts eNOS and caveolin‐1 expression and colocalization via glycocalyx degradation
Authors: Harding Ian , Mensah Solomon , Ebong Eno
Published: June 2021

Atherosclerosis is the main cause of cardiovascular diseases such as coronary artery and carotid artery disease and is known to occur in specific, atheroprone areas of the vasculature characterized by disturbed flow patterns. The endothelial glycocalyx, a mechanotransductive carbohydrate layer that lines the endothelium, helps maintain vascular homeostasis through flow‐induced regulation of vasculoprotective molecules such as nitric oxide. Areas of atherosclerosis development have been shown to contain diminished glycocalyx expression; however, it is unclear whether glycocalyx degradation contributes to atherosclerosis or is a result of its progression. Here, we investigated the effects of disturbed flow on glycocalyx‐mediated nitric oxide regulation in rat fat pad endothelial cells, which robustly express glycocalyx components. Using a custom parallel‐plate flow chamber, we found that disturbed flow (DF) patterns characteristic of atheroprone regions of the vasculature decrease glycocalyx coverage and thickness in comparison to uniform flow (UF). This decrease was accompanied by a reduction in endothelial nitric oxide synthase (eNOS) expression. For the first time, we discovered that DF also decreased the expression of caveolin‐1 (cav‐1), a core protein of caveolae microvesicles that assist in eNOS activation. Additionally, fluorescence microscopy also demonstrated decreased cav‐1 membrane and upstream distribution as well as decreased eNOS‐cav‐1 colocalization in areas of disturbed flow. Finally, UF exposure upon enzymatic degradation of heparan sulfate, a major glycocalyx component, resulted in DF‐like expression colocalization of eNOS and cav‐1, implicating the glycocalyx in eNOS and cav‐1 regulation. These findings provide new evidence to the body of information on glycocalyx‐mediated endothelial regulation, which collectively suggests that glycocalyx degradation may contribute to atherosclerosis development.

Support or Funding Information

This study was funded by National Institute of Health Grant K01 HL125499, National Science Foundation Grant DGE 1451070, Northeastern University Provost Start‐up funds, and an Engineering Dean's Fellowship provided by Northeastern University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does normobaric hypoxia sustain altitude acclimatization‐induced improvements in fluid intelligence during reintroduction to altitude?
Authors: Beidleman Beth A. , Sils Ingrid V. , Cymerman Allen , Fulco Charles S. , Staab Janet E. , Muza Stephen R.
Published: June 2021

This study examined whether normobaric hypoxia (NH)treatment is more efficacious than no treatment for sustaining high altitude (HA)acclimatization‐induced improvements in cognitive function during re‐introduction to altitude (RA) following 12‐day return to sea level (SL). Sixteen SL residents (age=23±6 yrs; mean±SE) participated in tests in the following order: 1) 4 d at SL, 2) 12 d at HA (4300 m), 3) 12 d at SL post HA acclimatization(Post) where each received either NH (n=9; FiO2=0.122) or Sham (n=8; FiO2=0.207)treatment, and 4) 24‐h re‐introduction to 4300 m (RA) in a hypobaric chamber. Cognitive performance was assessed using eight tests from the Automated Neuropsychological Assessment Metrics (ANAM) battery as measured by the number of correct responses per minute after 4 hours at SL, HA1, HA11, and RA. Simple reaction time (SRT) and SRT following cognitive fatigue (SRT2) were used to assess psychomotor performance. Go/no‐go (GNG) was used to assess attention and execution function Code substitution delayed (CDD) and Sternberg‐six letter memory (ST6) were used to assess long‐term memory (one component of crystallized intelligence). Procedural reaction time (PRT), mathematical processing (MTH), and spatial discrimination(SPD) were used to assess fluid intelligence. There were no differences in any measure of cognitive performance between the NH and Sham groups so data were combined. The PRT and MTH, respectively, decreased ~17% from SL (119±3; 34±3) to HA1 (99±5; 28±2) (P<0.05), then increased (P<0.05) at HA11 (114±4; 38±3), and remained unchanged at RA (113±3; 34±3). Similarly, SPD decreased ~14% from SL (44±3) to HA1 (38±1) (P<0.05) then increased (P<0.05)at HA11 (45±3), and remained unchanged at RA (45±3). SRT, SRT2, GNG, CDD, and ST6 were unchanged across any of the test days. These results demonstrate that psychomotor performance (SRT and SRT2), attention/executive function (GNG), and long‐term memory (CDD and ST6) were not impacted by acute or chronic exposure to 4300 m altitude. However, fluid intelligence, reflected in measures of processing speed (PRT), working memory(MTH), and spatial discrimination (SPD), decreased dramatically with acute exposure to 4300 m and improved with HA acclimatization. Moreover, improvements in fluid intelligence induced by HA‐acclimatization were retained during RA after 12 days at SL whether or not NH treatment was utilized. Authors' views not official U.S. Army or DoD policy.

Support or Funding Information

US Army Medical and Materiel Command

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Experimental Validation of a Respiratory Control Model
Authors: Diekman Casey O. , Thomas Peter J. , Wilson Christopher G.
Published: June 2021

Diekman et al. [Conf Proc IEEE Eng Med Biol Soc (2012) 2012:6669–72] introduced a model for closed‐loop respiratory control incorporating an explicit conductance‐based model of bursting pacemaker cells driven by hypoxia‐sensitive chemosensory feedback. Numerical solution of the model equations revealed two qualitatively distinct asymptotically stable dynamical behaviors: one analogous to regular breathing (eupnea), and a second analogous to pathologically rapid, shallow breathing (tachypnea). As an experimental test of the Diekman et al. model, we created a hybrid in vitro/in silico circuit. We used Real Time eXperimental Interface (RTXI) dynamic clamp to incorporate a living pacemaker cell recorded in vitro into a numerical simulation of the closed‐loop control model in real time. Here we show that the hybrid circuit can sustain the same bistable behavior as the purely computational model, and we assess the ability of the hybrid circuit to recover from simulated bouts of transient hypoxia.

Support or Funding Information

This work originated as an externally mentored postdoctoral project through the Mathematical Biosciences Institute [National Science Foundation (NSF) Grant DMS‐1440386] and was partially supported by NSF Grants DMS‐1413770 (P. J. Thomas), DMS‐1412877 (C. O. Diekman), and DMS‐155237 (C. O. Diekman).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Z‐band and M‐band Titin Splicing and Regulation by RBM20 in Striated Muscles
Authors: Rexiati Maimaiti , Chen Zhilong , Zhu Chaoqun , Cai Hanfang , Stern Allysa , Mozdziak Paul , Ge Ying , Ford Stephen P. , Nathanielsz Peter W. , Guo Wei
Published: June 2021

Titin (TTN) has multifunctional roles in sarcomere assembly, mechanosignaling transduction and muscle stiffness. TTN splicing generates variable protein sizes that are associated with its functions. Therefore, understanding TTN splicing is important to develop novel treatment for titin‐based diseases. The I‐band TTN splicing and regulation by RBM20 have been extensively studied. However, the Z‐ and Mband splicing and regulation remain poorly understood. Herein, we aimed to define the Z‐ and M‐band splicing in striated muscles, and also determine whether RBM20 regulates the Z‐ and M‐band splicing. Our data discovered four new Z‐band TTN splicing variants, and one of them dominates in mouse, rat, sheep and human heart. Frog and chicken hearts express only one variant. In skeletal muscles, three new Z repeats were detected, and variant with Zr4–6 skipping dominates in the fast muscles, whereas Zr4 skipping dominates in the slow muscle. No developmental changes were detected in the Z‐band. In the M‐band, two new variants were discovered with alternative 3′ splice site in exon363 (Mex5) and alternative 5′ splice site in intron362. However, only sheep heart expresses two new variants, while other species express only one variant with Mex5+. Skeletal muscles express all three M‐band variants with altered ratios of Mex5+ to Mex5−. Lastly, we revealed that RBM20 does not regulate the Z‐and M‐band splicing in the heart, but does in skeletal muscles. Taken together, we characterized the Z‐ and M‐band splicing and provided the first evidence of the role of RBM20 in the Z‐ and M‐band TTN splicing.

Support or Funding Information

This work was supported by the National Institute of Health/National Institute of General Medical Sciences [NIGMSP20GM103432]; the BGIA from the American Heart Association [16BGIA27790136 to WG]; the USDA National Institute of Food and Agriculture [Hatch project 1009266 to WG]; NICHD 1R01HD070096‐01A1 (SPF and PWN). Ying Ge would like to acknowledge NIH R01 HL109810 and HL096971.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Introgressive Hybridization in Hypoxia Adaptation of the Tibetan Mastiff
Authors: Signore Anthony , Yang Yingzhong , Storz Jay , Ge Ri‐Li
Published: June 2021

The role of introgressive hybridization as a source of adaptive genetic variation in animal populations is generally unclear, although recent findings suggest it may be more important than previously anticipated. While there are a number of tools to detect introgression, a major challenge is to experimentally confirm that it has played a role in adaptation by documenting the phenotypic effects of introgressed alleles. Animals native to high‐altitude are excellent models for such experimental studies, as it is often possible to formulate clear a priori hypotheses about the adaptive significance of particular changes in phenotype (for example, increases or decreases in the oxygen affinity of hemoglobin [Hb]). There is evidence to suggest that the high‐altitude adaptation of the Tibetan mastiff is, in part, the result of adaptive introgresssion with the Tibetan wolf. One of the introgressed regions between the Tibetan wolf and mastiff spans the entire β‐Hb gene cluster and the upstream cis‐regulatory elements. This has resulted in two non‐synonymous amino acid substitutions in the Tibetan mastiff, with respect to domestic dogs, in each of the adult expressed β‐type globin genes. To assess the functional effects of these mutations, we have measured the Hb‐O2 affinity of Tibetan wolves, Tibetan mastiffs and domestic dogs. These analyses revealed that the high‐altitude canids have Hb‐O2 affinity that is increased relative to domestic dogs (Figure 1). Thus, introgressive hybridization with the Tibetan wolf has resulted in a phenotypic change to the Tibetan mastiff that increases blood‐O2 saturation under hypoxia.

Support or Funding Information

This work is supported by the National Institutes of Health, the National Science foundation, and the Chinese Academy of Sciences.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantitative Method Development and Validation of Naloxone and 6α‐naloxol from Male African Green Monkey Plasma.
Authors: Bandaru Veera Venkata Ratnam , Vignola Justin N , Litvin Samantha R , Packer Amber M , Rizkallah Amy S , Orsini David A , Young Sheena A , Soni Sunil D , diTargiani Robert C , Pennington M Ross , Capacio Benedict R
Published: June 2021

Theopioid epidemic is a rapidly growing crisis around the world. Current models forpharmacokinetic assessment of countermeasures use African green monkeys (AGM). Arapid, sensitive and selective method was developed and validated usingLC‐MS/MS for the determination of naloxone and 6α‐naloxol in AGM plasma. Samplepreparation involved a simple solid phase extraction (SPE) using ahydrophilic‐lipophilic‐balanced reversed‐phase cartridge. Separation of naloxoneand 6α‐naloxol was achieved on a Halo C18, 2.1 × 50 mm, 2.7μ analytical columnusing an Agilent 1290 Infinity HPLC. A solvent system of methanol‐water‐formicacid was used for the elution of analytes. The analytes were detected and monitoredby a SCIEX QTRAP 6500+ triple quadrupole tandem mass spectrometer operatedin electrospray ionization (ESI) positive mode and multiple reaction monitoring(MRM). Molecular ion transitions for naloxone, 6α‐naloxol and naloxone‐D5 were 328.0/212.0,330.0/185.0, and 333.1/212.0 m/z, respectively. The method was validated usingcalibration and recovery assays with spiked samples. Linear calibration curveswere generated over the range of 1.5–100 ng/ml with values for the coefficientof determination (R2) of >0.9950. The values for both inter‐dayand intra‐day precision and accuracy were well within the generally acceptedcriteria for analytical methods (≤15%). This method was subsequentlyused to measure plasma concentrations of naloxone and 6α‐naloxol in male AGMs administerednaloxone subcutaneously and intravenously. The developed method demonstratedexcellent recovery, repeatability, accuracy and sensitivity.

Support or Funding Information

DISCLAIMERS. The views expressed in this abstractare those of the authors (s) and do not reflect the official policy of theDepartment of Army, Department of Defense, or U. S. Government. This researchwas supported by the Defense Threat Reduction Agency – Joint Science andTechnology office, Medical S&T Division.

Thisproject was supported in part by an appointment to the Research ParticipationProgram for the U.S. Army Medical Research and Materiel Command administratedby the Oak Ridge Institute for Science and Education through an agreementbetween the U.S. Army Medical Research and Materiel Command.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lymphocyte Specific Protein‐1 Suppresses Hepatocarcinogenesis Driven by Mutant β‐catenin and Met Overexpression
Authors: Koral Kelly , Tao Junyan , Bhushan Bharat , Stoops John , Mars Wendy , Monga Satdarshan Pal , Michalopoulos George
Published: June 2021

Lymphocyte specific protein‐1 is the gene with the most copy number variations in a cohort of human hepatocellular carcinoma cases. Previous studies have demonstrated that LSP‐1 acts as a suppressor of hepatocellular proliferation in both cancer cell lines and during liver regeneration after partial hepatectomy as well as an inhibitor of migration. Additionally, concurrent Met overexpression and mutant β‐catenin expression has been demonstrated in 9–12.5% of human HCC cases. Therefore, in the present study, we evaluated whether LSP1 expression affects hepatocarcinogenesis driven by mutant β‐catenin (S45Y) and hMet overexpression by utilizing the sleeping beauty transposase system with hydrodynamic tail vein injections in LSP‐1 transgenic (TG) and WT C57/Bl6 mouse models. LSP1 TG mice overexpress LSP1 specifically in hepatocytes using the albumin promoter, alpha‐fetal protein enhancer. LSP1 TG mice displayed very little histological changes as well as decreased MAPK activation in comparison to the C57 WT mice, which exhibited macroscopic tumors, large PCNA positive proliferating nodules and increased ERK phosphorylation at 7 weeks post injection. Our results demonstrate that expression of LSP1 suppresses mutant β‐catenin and Met driven HCC and may represent a novel therapeutic option for this subset of HCC.

Support or Funding Information

Supported by Department of Defense grant CA160119, the University of Pittsburgh Menten Endowment, and the Cleveland Foundation Morningstar Fund.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Immune trade‐offs shape disease outcomes at individual and population scales
Authors: Ezenwa Vanessa
Published: June 2021

Most aspects of an animal's life history are subject to trade‐offs, where a beneficial change in one trait is linked to a detrimental change in another. Immune function is no different and trade‐offs between different components of the immune system are well described. Importantly, these trade‐offs may have significant implications for infectious disease outcomes. For example, trade‐offs in immunity may be particularly important during concurrent infection (i.e. co‐infection), if the host immune response to one pathogen negatively affects the response to another. Focusing on trade‐offs between the T helper cell 1 (Th1) and Th2 branches of the immune system, we examined how infection with gastrointestinal worms, which trigger a Th2‐type immune response in the host, affects the immune response to a bacterial pathogen (Mycobacterium bovis, causative agent of bovine tuberculosis) for which a Th1‐type response is required for defense. By integrating experimental and longitudinal studies of a wild population of African buffalo (Syncerus caffer), we show that this well‐known trade‐off in immune function operates on both ecological and evolutionary time scales with repercussions for individual disease severity and population‐level disease dynamics.

Support or Funding Information

National Science Foundation (Award #DEB‐1102493)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Do Student Misconceptions about Mutations Persist throughout College Education?
Authors: Couch Brock , Seipelt‐Thiemann Rebecca , Reid Joshua , Grimes Zachary , Wasendrof Chloe , James Olena , Peters Nick , Boury Nancy
Published: June 2021

Genetics is a widely discussed topic both within and outside of science. One particular topic in genetics that is readily encountered outside of the science classroom is mutations. Students encounter mutations through several different avenues (e.g., news, comic books, movies) that may not be scientifically correct. This pre‐exposure to genetics concepts has the potential to misinform their ideas before ever enrolling in a genetics course. Because of this misinformation, genetics can be difficult for students because of the inconsistences in complex terminology, abstract concepts, and having to think across multiple scales. In order for instructors to create curricula for students, they first need to understand the misconceptions of genetics students may bring into the classroom. Instructors also need to understand if their practice is allowing students to overcome their misconceptions or if they are persistent throughout the students' education. The aim of this project is to 1) understand students' misconceptions about mutations and 2) determine if these misconceptions persist as student progress through their undergraduate education. A draft mutations concept inventory was used to collect responses from 454 students in introductory and advanced genetics courses, as well as introductory biology and advanced microbiology courses. The multiple choice questions included a correct answer and distractors from known misconceptions. Utilizing non‐metric dimensional scaling, data was visualized to determine which misconceptions persisted across different courses and which were resolved or created. Further studies aim to more fully explore conceptual understanding and specific misconception types by student group and so that instructors can provide resources that will enable students to gain an accurate conceptual understanding of mutations within genetics, microbiology, and molecular biology.

Support or Funding Information

NSF Award #1710262

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.