bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Chromosome 14 in the Genetic Basis for Endurance Exercise Capacity and Responses to Training in Mice
Authors: Massett Michael P. , Avila Joshua J. , Kim Seung Kyum
Published: June 2021

A significant quantitative trait locus (QTL) for exercise capacity and a suggestive QTL for the change in exercise capacity were identified previously on mouse Chromosome 14 (Chr 14). The aim of this study was to investigate the role of Chr 14 in endurance exercise capacity and responses to training in mice. Exercise capacity and training responses were measured in chromosome substitution strain (CSS) mice derived from PWD/PhJ (PWD) and C57BL/6J (B6) inbred strains, denoted as B6.PWD14. These mice carry Chr 14 from the PWD donor inbred strain on the genetic background of a host B6 inbred strain. Twelve‐week‐old male and female mice from each strain (B6, PWD, B6.PWD14) were assigned to sedentary control (SED) or exercise training (EX) groups (n=6/group/sex). All mice completed a graded exercise test before and after the exercise‐training period to determine endurance exercise capacity. Responses to training were calculated from the difference in exercise capacity between post‐ and pre‐training graded exercise tests. EX mice completed a 4‐week training program consisting of treadmill running 5 days/week, 60 min/day at a final intensity equivalent to approximately 65% of the maximal work‐load (speed and incline) attained during the graded exercise test. Overall, there were significant effects of sex and strain on body mass and exercise capacity (pre and post), but only a significant effect of sex on responses to training in EX mice. In male mice, there were no significant differences in endurance exercise capacity or responses to training between B6 and B6.PWD14 mice. In contrast, female B6.PWD14 mice had higher pre‐ and post‐training endurance exercise capacity than female B6 mice. However, there were no significant differences between mice from these strains for responses to training. These data demonstrate that PWD alleles on Chr 14 significantly affect pre‐ and post‐training endurance exercise capacity in a sex‐specific manner. Furthermore, these data suggest that Chr 14 contains genetic factors influencing endurance exercise capacity, but not the responses to training in these strains of mice.

Support or Funding Information

Supported by NIH R01 HL085918, APS William Townsend Porter Pre‐doctoral Fellowship (JA), and Texas A & M University Institute for Advanced Study (TIAS).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acute Effects of Aerobic Exercise on Arterial Stiffness and Wave Reflection in Young Men and Young Premenopausal Women
Authors: Lim Jisok , Kim Han‐Kyul , Hwang Chueh‐Lung , Yoo Jeung‐Ki , Perez Hannah , Handberg Eileen M. , Christou Demetra
Published: June 2021

Arterial stiffness and wave reflection are associated with increased risk for cardiovascular disease (CVD), while aerobic exercise is known to decrease CVD risk. The effects of chronic aerobic exercise training on arterial stiffness and wave reflection have been extensively studied, but data on the acute effects of aerobic exercise on these measures are primarily based on men and are limited and conflicting. The purpose of this study was to examine the sex‐specific acute vascular responses to high‐intensity interval training (HIIT), moderate‐intensity continuous training (MICT) and low‐intensity continuous training (LICT) in young adults. Thirty healthy adults, 15 young men and 15 premenopausal women (21.5±0.4 vs. 21.4±0.7 years of age, means ± SE, P =0.9) participated in this randomized crossover study. The SphygmoCor Xcel device (AtCor medical) was employed to assess arterial stiffness using carotid to femoral pulse wave velocity (cfPWV) and wave reflection using augmentation index (AIx). Measures of cfPWV and AIx were obtained prior to exercise (PreE), following the end of a single bout of HIIT, MICT and LICT on the treadmill (PostE), and following recovery from exercise. HIIT consisted of 4 × 4‐min intervals at 90% peak heart rate (HRpeak) interspersed by recovery bouts at 70% HRpeak for a total of 40 min, whereas MICT and LICT consisted of 47 min at 70% and 50% HRpeak, respectively. cfPWV remained unchanged in response to a single bout of exercise, regardless of exercise intensity or sex (5.5±0.1 vs. 5.5±0.1 vs. 5.4±0.1 m/s, PreE vs. PostE vs. recovery; P=0.2 for main effect of time). AIx decreased following recovery compared with PreE and PostE (P≤0.003), but this response was not influenced by exercise intensity or sex (10.3±1.7 vs. 9.0±1.7 vs. 5.0±1.5 %, PreE vs. PostE vs. recovery; P<0.0001 for main effect of time; P=0.8 for sex × exercise × time). In conclusion, exercise intensity and sex do not modulate central arterial stiffness and wave reflection following a single bout of aerobic exercise. Central arterial stiffness is unaffected by acute exercise, whereas wave reflection decreases following recovery from exercise.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aldosterone‐sensitive HSD2 neurons in the nucleus of the solitary tract: gene expression and axonal projections in mice
Authors: Geerling Joel C. , Resch Jon M. , Lowell Brad B. , Peltekian Lila , Narayan Sowmya V.
Published: June 2021

Dietary sodium deficiency elevates aldosterone, which in turn stimulates a unique group of neurons in the nucleus of the solitary tract (NTS). In the adult mouse brain, these NTS neurons alone express the enzyme 11‐beta‐hydroxysteroid dehydrogenase type 2 (HSD2), a hallmark of aldosterone‐sensitive cells. Here, we add detailed information about HSD2 neurons in mice, which initially were characterized in rats. First, we confirm that HSD2‐immunoreactive neurons in mice are found exclusively in the NTS. Next, using in situ hybridization, we show that these neurons robustly express Hsd11b2, the mineralocorticoid receptor (Nr3c2), vesicular glutamate transporter 2 (Slc17a6), the transcription factor Phox2b, and the angiotensin II receptor 1a (Agtr1a). Conversely, they do not express protein markers for cholinergic and monoaminergic neurons or mRNA transcripts for the peptides galanin (Gal), dynorphin (Pdyn), or angiotensinogen (Agt), which are abundant in the surrounding NTS. Next, in Hsd11b2‐Cre mice, we use Cre‐dependent axon labeling to refine conventional axon tracing data from rats. HSD2 neurons project their axons into the ipsilateral reticular formation, where they course rostrally past the trigeminal motor nucleus before turning dorsally towards the parabrachial complex (PB). There, the axons form dense terminal fields in the pre‐locus coeruleus (pLC) and an adjoining part of the medial PB subnucleus and arborize laterally in a subregion of the central lateral PB subnucleus. In addition, some axons course rostrally through the midbrain reticular formation, into the medial forebrain bundle, and ultimately form a dense terminal field in a subregion of the ventrolateral bed nucleus of the stria terminalis (BSTvL) that is homologous to the rat fusiform subnucleus. En route to the BSTvL, small numbers of axons branch and form boutons in the parasubthalamic nucleus, suprafornical lateral hypothalamic area, central nucleus of the amygdala, and paraventricular hypothalamic nucleus. Finally, using dual retrograde tracing, we found that projections to PB and BSTvL originate from separate subsets of HSD2 neurons. While their long‐range projection to BSTvL likely promotes sodium appetite, their axonal projections to PB and other sites may alter the taste of sodium and mediate other behavioral correlates of sodium depletion such as nausea, anhedonia, or fatigue.

Support or Funding Information

K08 NS099425 (JCG); Aging, Mind, and Brain Institute, University of Iowa (JCG); F32 DK103387 (JMR); R01 DK075632, R01 DK096010, R01 DK089044, R01 DK111401, P30 DK046200, P30 DK057521 (BBL)

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 influx at hyperpolarized membrane potentials modulates depolarization‐triggered exocytosis in bovine chromaffin cells.
Authors: Fomina Alla F. , Yang Lukun
Published: June 2021

Perturbations in Ca2+ entry via neuronal transient receptor potential (TRP) and ORAI family proteins have been shown to alter synaptic transmission, and suggested to play a role in several neurodegenerative diseases including Huntington's, Alzheimer's and Parkinson's diseases. Specific mechanisms by which of TRP/ORAI channels modulate synaptic signaling are not completely understood. To determine the role of Ca2+ influx trough non‐voltage gated Ca2+ channels in regulation of neurotransmitter release, we investigated effects of modulation of Ca2+ influx at hyperpolarized voltages on exocytosis in bovine chromaffin cells using a combination of imaging and electrophysiological techniques. We found that a small inwardly directed membrane current whose amplitude was dependent on the concentration of extracellular Ca2+ and Na+, was present at resting membrane potential. Augmentation of this current either by hyperpolarization (from −50 mV to −90 mV) or stimulation with bradykinin (1 μM), a catecholamine secretagogue, elevated cytosolic Ca2+ concentration at hyperpolarized potentials and facilitated exocytotic responses triggered by short depolarizations (<500 ms in duration). Exocytotic responses triggered by long depolarizations (>500 ms in duration) were suppressed at hyperpolarized holding potentials. Reversal of Na+/Ca2+ exchanger by removal of Na+ from bath solution did not cause depolarization‐independent exocytosis at −50 mV holding potential. Membrane hyperpolarization from −50 mV to −90 mV in the absence of direct Na+/Ca2+ exchanger activity evoked depolarization‐independent exocytosis. Our findings provide first experimental evidence that in neuroendocrine cells perturbation in Ca2+ influx at negative voltages with physiologically relevant stimuli significantly modulates exocytotic responses to depolarization‐triggered Ca2+ entry via voltage‐gated Ca2+ channels, and that Ca2+ influx via native Ca2+ channels operating at negative voltages is functionally coupled with Na+/Ca2+ exchanger.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Organismal Sex is a Major Determinant of Phenotypic and Molecular Changes Caused by Genetic Inactivation of the Mitochondria‐specific Deacetylase SIRT3
Authors: Roos Carolyn M. , Hagler Michael A. , Zhang Bin , Fujimoto Hirokazu , Miller Jordan D.
Published: June 2021

Sirtuin 3 (SIRT3)—a deacetylase localized to the mitochondria—deacetylates a number of mitochondria‐specific proteins, including the mitochondrial antioxidant enzyme MnSOD. We previously reported that genetic inactivation of SIRT3 impairs endothelial function and increases pro‐osteogenic markers in early stages of atherosclerosis. While recent data suggest that the molecular and phenotypic effects of SIRT enzyme inactivation can act in a highly sex‐dependent manner, we have not systematically studied sex differences in our unique mouse strains. Here, we report our first effort to explore the presence of molecular and phenotypic sex differences following inactivation of SIRT3 in hypercholesterolemic mice. We used female and male Ldlr−/−/ApoB100/100 mice that were either wild‐type (LA‐SIRT3+/+) or null for SIRT3 (LA‐SIRT3−/−). Following 3 months of western diet feeding, we assessed endothelial function of aorta using isolated organ bath chambers and measured changes in gene expression using qRT‐PCR. We did not observe sex differences at baseline in hypercholesterolemic mice, as endothelium‐dependent relaxation to acetylcholine (ACH) was similar between female LA‐SIRT3+/+ and their male LA‐SIRT3+/+ littermates (61.1%±2.2, 57.9%±2.7; respectively). Interestingly, genetic inactivation of SIRT3 did not impair responses to ACH in female LA‐SIRT3−/− mice (61.1%±4.2), whereas responses to ACH were modestly impaired in male LA‐SIRT3−/− mice (53.9%±1.9). Consistent with SIRT3's role as a post‐translational protein modifier, expression of MnSOD was unchanged across all sexes and genotypes. Expression of genes related to pathophysiological changes in atherosclerotic plaques did change in a highly sex‐dependent manner, however, as osterix gene expression levels (related to plaque calcification) were markedly increased in male LA‐SIRT3−/− compared to their sex‐matched wild‐type littermates and to their female counterparts. Surprisingly, while female LA‐SIRT3−/− mice showed dramatic increases in expression of interleukin 6 (related to inflammation) compared to their wild‐type littermates, male LA‐SIRT3−/− had robust reductions in expression in interleukin 6 compared to their LA‐SIRT3+/+ littermates. Collectively, these data suggest there are significant, sex‐dependent molecular and phenotypic effects of SIRT3 inactivation in hypercholesterolemic mice, which may also drive a paradoxical dissociation between pro‐osteogenic and pro‐inflammatory signaling under conditions of mitochondrial dysfunction.

Support or Funding Information

NHLBI R01‐HL111121

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phrenic afferent projections to the cervical spinal cord in spinal intact and spinal injured rats
Authors: Turner Sara , Schuster Cassandra , Fusco Anna , Cotto Alejandra , Pignataro Megan , Sunshine Michael , Fuller David
Published: June 2021

The phrenic nerve contains both myelinated and unmyelinated afferent projections. Relatively little is known, however, regarding the spinal distribution of these sensory projections. Accordingly, our first goal was to create a map of phrenic afferent projections throughout the cervical spinal cord. Afferents were traced via prolonged phrenic nerve soak with Cascade Blue (CB) in adult Sprague‐Dawley rats. Spinal tissues were harvested, sectioned (40μm) from C2–C7, and processed with routine immunochemical methods. Extensive labeling of afferent projections was observed throughout the ipsilateral dorsal horn (laminae II and III) and intermediate grey region (laminae IV and V) in segments C2–C6. Phrenic afferent projections were crossed the spinal midline in 13% of the tissue sections, and were occasionally observed to reach the contralateral dorsal horn (laminae III). Robust phrenic motoneuron (PhrMN) labeling was observed from C3–C5, and PhrMN dendrites projected across the spinal midline in 30% of tissue sections from C3–C5. Our second objective was to determine if high cervical spinal cord injury (SCI) induced anatomical plasticity in the phrenic sensorimotor system. The phrenic nerve was traced using the same CB method in Sprague‐Dawley rats at 12 weeks following lateral hemisection of the C2 spinal cord. Preliminary analysis indicates that following SCI, phrenic afferents and PhrMN dendrites project across the spinal midline at approximately the same frequency as in spinal‐intact rats (p>0.36). However, we noted that the likelihood of observing phrenic afferent projections extending to the region of the ipsilateral phrenic motor nucleus (laminae VIII‐IX) was increased after SCI (p<0.05). By combining CB labeling with Choline acetyltransferase (ChAT) immunostaining, we verified the presence of laminae VIII‐IX phrenic afferents by observing CB‐positive and ChAT negative fibers. We conclude that phrenic sensory afferent projections 1) are prominent in the ipsilateral C3–C5 dorsal horn and intermediate grey region, 2) can project to the contralateral C3–C5 dorsal horn, and 3) undergo remodeling after high cervical SCI.

Support or Funding Information

SPARC OT2 OD023854 (DDF), 1 R01 NS080180‐01A1 (DDF)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Brains to Inspire Minds: A Unique Outreach Collaboration with the Boston Museum of Science
Authors: Babcock Katharine Jane , Kong Elizabeth , Zumwalt Ann C.
Published: June 2021

The Department of Anatomy and Neurobiology at Boston University School of Medicine has recently undertaken an exciting collaboration with the Hall of Human Life (HHL) at the Museum of Science, Boston to engage the general public in a dialogue about the human brain. This partnership is unique in that the scientists and the museum staff work together to develop demonstrations that neither group would have the resources to produce on their own. In this initiative we use post‐mortem human brains to get the attention of museum visitors and pique their curiosity. Visitors are encouraged to ask questions and drive discussions based on their own interests, while educators adapt their language as needed when interacting with audiences of differing age groups. Topics vary from how information is relayed to and from the brain, to specific interests such as recent public health concerns surrounding concussion in sports. In addition to human tissue, the exhibit also includes human anatomical models and dissections of simpler nervous systems (e.g. rat, mouse, gerbil, fish, and fruit fly) to stimulate comparative and functional neuroanatomical discussions. The main objective is to cultivate a broad‐based, easily accessible learning experience that mirrors the HHL's mission to “inspire all who enter to think, learn, and question.”

Support or Funding Information

AAA Education Outreach 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.
Positive Impact of an Interactive 3D Neuroanatomy E‐learning Resource on Students' Spatial Neuroanatomical Knowledge
Authors: Allen Lauren , Wright Trinette , Eagleson Roy , de Ribaupierre Sandrine
Published: June 2021

Neuroanatomy is one of the most challenging topics in anatomy for many novice students, who report it to be the most difficult subject in their anatomy curriculum. A primary reason for the perceived difficulties is the high degree of spatial complexity that exists between structures. The development of interactive three‐dimensional (3D) models in neuroanatomy curricula may provide an effective solution for alleviating difficulties students encountered with two‐dimensional (2D) learning resources, however this has yet to be fully examined in the literature.

Interactive 3D and 2D e‐learning resources, as well as a novel virtual syncretion assessment tool, were developed to complement undergraduate anatomy instruction. The virtual syncretion assessment required participants to place neuroanatomical structures in a partial 3D neuroanatomical model. This assessment format allowed for the evaluation of spatial knowledge based on the accuracy of the 3D placement of structures rather than conventional nominal responses. Performance on the virtual syncretion assessment was measured by both the selection of correct structures, and the accuracy of the placement of structures within the partial 3D neuroanatomical model.

One hundred seventy‐three participants completed the study, which utilized a cross‐over design to separate participants into two groups. Each group initially completed an anatomy knowledge pretest, followed by access to either the 3D or 2D neuroanatomy e‐learning resource. Participants completed both an anatomy knowledge quiz and virtual syncretion assessment prior to utilizing the second learning resource. A second anatomy knowledge quiz and virtual syncretion assessment were administered following participants' exposure to the second e‐learning resource.

Anatomy knowledge quiz scores of participants who initially accessed the 3D learning resource increased significantly more than the students who initially accessed the 2D e‐learning module. Participants who initially utilized the 3D e‐learning resource performed significantly better on the digital syncretion assessment than participants who initially utilized the 2D e‐learning resource. Additionally, participants who accessed the 3D e‐learning resource subsequent to the 2D e‐learning resource significantly improved their performance on the final virtual syncretion assessment. No significant improvement in performance was observed for participants who accessed the 2D e‐learning resource subsequent to the 3D e‐learning resource. Results of this study could be used to inform the effective development and deployment of interactive 3D e‐learning resources to improve neuroanatomy instruction and assessment.

Support or Funding Information

Social Sciences and Humanities Research Council

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of Blood Cell Populations in the Development and Regeneration of a Colonial Ascidian
Authors: Boyd Megan , Balde Erick , Keeling Elena
Published: June 2021

Botrylloides violaceus is a colonial marine invertebrate with the capability of regenerating its whole body when only blood cells and vessels are present. These tunicates are more closely related to vertebrates than other model organisms that undergo whole‐body regeneration. Regeneration is induced when the organism sustains an injury and is carried out by a circulating stem cell population. Botrylloides’ sister genus, Botryllus, also has the capability to regenerate; however, regeneration in Botrylloides occurs more readily. We investigated whether stem cell abundance in the blood differs across life stages of B. violaceus and between the two genera. Using brightfield microscopy, we quantified blood cell types from different life stages and from regenerating animals, emphasizing the hemoblasts which include the stem cell population. Preliminary data show a significant decrease in hemoblasts between adult and young Botrylloides, a decrease and then plateau during the regeneration process, and significantly fewer hemoblasts in adult Botryllus than adult Botrylloides. Our data suggest that a high number of stem cells within circulating blood may be important for inducing regeneration. The decrease in frequency in regenerating animals may imply that stem cells are partially depleted during regeneration.

Support or Funding Information

California Polytechnic State University, Biological Sciences Department

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 assessment of interaction between eNOS and Alpha hemoglobin by Bio‐layer Interferometry
Authors: Ma Dongying , Ackerman Hans
Published: June 2021

Alpha globin, a subunit of red blood cell hemoglobin, is also expressed in human and mouse resistance artery endothelial cells. Qualitative protein‐protein interaction methods have been used to identify a macromolecular complex that forms between alpha globin and endothelial nitric oxide synthase (eNOS). The aim of this study is to characterize the interaction between alpha globin and eNOS quantitatively using bio‐layer interferometry (BLI). To this end, alpha globin was purified from whole hemoglobin using ion exchange chromatography and the purity was assessed by mass spectrometry. The oxygenase of eNOS (eNOSox) was expressed in PGEX vector and UT5600 E. coli system. Further, the ForteBio Octet system was used to identify the binding characterization of purified alpha globin with eNOS, with its molecular chaperon a‐Hemoglobin stabilizing protein (AHSP) as control. The results showed that the affinity constant KD between alpha globin and AHSP is 1.09×10−7 M, which was calculated using the association rate ka (2.28×104 M−1s−1) and dissociation rate (2.49×10−3 s−1). The affinity constant KD between alpha globin and eNOSox is 1.31×10−6 M, which was calculated using the association rate ka (1.16×103 M−1s−1) and dissociation rate (1.51×10−3 s−1). Smaller affinity constant indicates greater affinity of analyte to ligand. Thus, the interaction between Alpha globin and AHSP is tighter than the interaction between alpha globin and eNOSox. However, the lower kd value for alpha‐globin and eNOSox indicates the slower dissociation, compared to dissociation from AHSP. This result will help us to better understand the molecular mechanisms of the regulation of alpha globin on eNOS function, and provide foundation for the development of chemical drug compounds which target specifically on this interaction.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical Characterization of MarR and TetR Proteins from Clostridium difficile
Authors: Takahashi Rei , Panchal Bhumika , Kim Da Eun , Wilkinson Steven
Published: June 2021

Clostridium difficile, the causative agent of pseudomembranous colitis, is a leading cause of hospital acquired infections in the United States with approximately 420,000 – 700,000 new infections each year. Of major concern is the increasing rate of patient mortality and the rise in C. difficile infections in traditionally low‐risk groups (e.g. otherwise healthy adults) marked by the emergence of hypervirulent strains characterized by increased resistance to a range of antibiotics. In this study, we report initial efforts to purify and characterize MarR (multiple antibiotic resistance regulator) and TetR (tetracycline resistance regulator) proteins from C. difficile as part of a larger investigation into the possible roles of these proteins in the antibiotic resistance mechanisms of this pathogen. MarR and TetR proteins are known to regulate genes required for antibiotic resistance in a number bacterial species but their functions in C. difficile remain unknown. A common feature of members of these two protein families is the specific binding of small‐molecule ligand(s) with accompanying changes in protein conformation and altered DNA‐binding affinity. Here we report initial results from analytical size exclusion to characterize the oligomerization states of C. difficile MarR and TetR proteins and results from biochemical analyses to characterize DNA‐binding. We also present results obtained from protein homology modeling with a particular focus on the conservation of residues in potential ligand‐binding sites and predicted DNA binding domains.

Support or Funding Information

California Polytechnical State University Research, Scholarly and Creative Activities 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.
Deciphering the effect of salts on Bilirubin Oxidases activity
Authors: Roussarie Elodie , Bichon Sabrina , Perrière Guy , Mano Nicolas , Stines‐Chaumeil Claire
Published: June 2021

Bilirubin Oxidases (BODs) and laccases belong to the Blue Multicopper Oxidases family (MCOs). Both enzymes couple the oxidation of substrates to the reduction of O2 to H2O. In contrast to laccases, BODs display high activity and stability at neutral pH and are more tolerant to chloride [1]. These unique features make BODs useful in a large number of biotechnological applications such as biosensors for jaundice detection, dyes degradation, and as a cathodic enzyme in enzymatic biofuel cells [2]. Glucose/oxygen enzymatic biofuel cells use the energy of redox reactions catalyzed by enzymes to produce electrical energy. Glucose oxidizing enzymes such as Glucose Oxidase (GOX) and oxygen reducing enzymes such as BODs are used at the anode and at the cathode respectively. Such biofuel cells are good candidates to be implanted in the human body of diabetic people and would be the only in vivo power source able to power glucose sensors. To be implanted, cathodic and anodic enzymes have to be enough stable and resistant to physiological salts to be efficient several weeks or months in biological fluids.

Here, I will focus my presentation on the enzymatic cathodic part. BODs and laccases are well known to be sensitive to high salt concentrations [3] and their stability on the electrode needs to be improved. Efforts in this direction allowed our team to identify the BOD from Bacillus pumilus with interesting features as a cathodic enzyme [4]. By using a sequence analysis approach, a new BOD was identified with very interesting features compared to other BODs which makes it of great interest to be used as a cathodic enzyme in biofuel cells.

Particularly, in contrast to most BODs described, this new BOD is not inhibited but activated by several salts (up to 1,7 times). This characteristic makes this BOD very interesting considering the fact that all biological fluids contain relatively high levels of salts (such as 140mM NaCl in the blood for example). For this reason, we have compared the effect of various salts and their mechanisms on the enzymatic activity of BOD from Bacillus pumilus and the new BOD in solution and immobilized on electrode. Molecular modeling studies were also performed on both enzymes to decipher the mechanisms of activation/inhibition of different salts and more precisely the sodium chloride effects at the atomic level.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the mechanism of 6‐hydroxynicotinate 3‐monooxygenase (NicC), an enzyme involved in nicotinate degradation
Authors: Nakamoto Kent , Gerislioglu Selim , Snider Mark J.
Published: June 2021

N‐Heterocyclic aromatic compounds (NHACs) are employed in daily life and are environmental contaminants. Due to NHAC's toxic, mutagenic, and carcinogenic effects, improved purification methods are imperative. Understanding bacterial degradation of NHACs may assist future bioremediation techniques. Nicotinic acid (NA) degradation serves as a good model for NHAC catabolism. 6‐Hydroxynicotinate‐3‐monooxygenase (NicC) is an enzyme involved in the degradation of NA. NicC catalyzes the hydroxylative decarboxylation of 6‐hydroxynicotinic acid (6‐HNA) and NADH to 2,5‐dihydroxypyridine (2,5‐DHP) and NAD. Understanding the mechanism of this reaction, which activates the pyridine of NA for ring‐opening, will provide greater insight on the conditions NHAC degradation require in bacteria. This knowledge could result in the optimization of bioremediation efforts that are already known and reduce the amount of hazardous waste in the environment. Two mechanisms for the oxidation half‐reaction of NicC have been proposed. One mechanism (A) predicts the formation of a covalent complex between NicC and 6‐HNA, decarboxylation, and subsequent hydroxylation by hydroperoxyflavin. A second mechanism (B) proposes a base deprotonates 6‐HNA leading to hydroxylation by hydroperoxyflavin and then decarboxylation. The research objective is to elucidate the mechanism of this reaction. Current results suggest NicC catalyzes oxidative decarboxylation by mechanism B. ESI‐MS data indicate no evidence of a covalent complex between the enzyme and the substrate, as the protein mass was equivalent to only the enzyme and not the ES complex. Mechanism A involves the keto tautomer requiring nitrogen to be present in the aromatic ring, while mechanism B has no such requirement. To test this, NicC was shown to catalyze the reaction of 4HBA, a homocyclic analog of 6‐HNA. This result confirms that NicC doesn't require nitrogen in the ring and disproves mechanism A. The coupling of the WT NicC reaction was determined to generate one NAD for every 2,5‐DHP with a time point HPLC assay. Future work will compare the coupling and rates of the WT NicC reaction to those of the variant NicC reactions to identify the key catalytic residues.

Support or Funding Information

Henry J. Copeland Fund for Independent Study at the College of Wooster

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Purification of Acid Phosphatase from Lupinus albus for Characterization
Authors: Phillips Sophia R. , Fogle Emily J.
Published: June 2021

The phosphorus cycle is an important biogeochemical process. Part of the phosphorus cycle occurs in soils where organic compounds containing phosphorus are converted into more bioavailable forms. However, the interactions between phosphatases that catalyze these reactions in soils are not well‐characterized. This research aims to characterize the adsorption and enzymatic activity of the adsorbed enzyme in different soil compositions. Thus far, research has been focused on overexpressing an acid phosphatase from Lupinus albus that is known to be excreted into soils and purifying the protein. Overexpression has been analyzed using SDS‐PAGE gels and UV‐Vis spectroscopy assays for enzyme activity. To date, overexpression of the enzyme is poor and further research will involve experimentation with an acid phosphatase gene from another source, Aspergillus ficuum.

Support or Funding Information

Frost Undergraduate Student Summer Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Notch Heterodimer NICD Complexes have Divergent Functions Compared to NICD Homodimer Complexes
Authors: Crow Jacob Jeffery , Albig Allan
Published: June 2021

The mammalian Notch signaling system consists of four receptors that can be activated by five membrane bound ligands on adjacent cells. Activation of Notch receptors results in the proteolytic release of Notch intracellular domains (NICD) that translocate to the nucleus to initiate transcriptional programs. Individual N1ICD molecules have been shown to homodimerize on DNA, however a number of basic questions remain regarding dimerization of NICD domains. Based on conserved sequences and structural similarities between NICD domains, we hypothesized that various NICD domains may be able to function as heterodimer complexes and that these heterodimeric complexes may exhibit different transcriptional activities compared to their homodimer counterparts. To address this hypothesis, we devised a co‐immunoprecipitation strategy to examine NICD dimerization in vivo. Our preliminary results show for the first time that some, but not all, NICDs are capable of functioning as heterodimers, even though all NICDs are capable of forming homodimers. This observation, together with the fact that individual cells often express multiple Notch receptors, prompted us to compare the transcriptional activities of homo‐ vs hetero‐dimer complexes. Using a Notch responsive luciferase reporter assay, we determined that NICD heterodimers have transcriptionally divergent activities compared to their NICD homodimer counterparts. Together, these and other observations suggest that signaling in the Notch system does not function solely on a homodimer basis. Instead, and more akin to other signaling systems, our results suggest that Notch signaling will also be influenced by NICD heterodimer complexes.

Support or Funding Information

NIH Grant 2R15GM102852‐02

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improving Student/Faculty Engagement: Impact of End of Lecture Classroom Surveys on Student Evaluations and Motivation
Authors: Miller Keith R.
Published: June 2021

Student success is directly tied to effective feedback criteria where the focus is on what is being learnt and how students should go about it. To provide this learning environment, it is best if the student initiates the feedback conversations. From Fall 2015–2017, end of lecture surveys were included as part of each class session for an introduction to biochemistry class for junior and senior undergraduates. In this teaching strategy, students were anonymously asked to describe aspects of the day's lecture that were (a) surprising, (b) interesting or useful, and (c) confusing on a form turned in at the end of class. While submissions could remain anonymous, students were encouraged to place their name on the form for direct email feedback after class to answer any questions they had. These comments would then be used as a springboard for subsequent lectures, resulting in an immediate impact on student engagement and the clarity of the lectures. At the end of the semester, students were surveyed anonymously regarding whether this teaching strategy assisted in their learning for the course, if they chose to receive email feedback, and if they would like other courses to use these surveys. In the Fall 2014 semester, before these classroom surveys were initiated in the introduction to biochemistry class, most of the students did not feel the subject matter was understandable, half did not feel motivated to learn, and half did not feel the professor had created an atmosphere of mutual respect. After implementation, in Fall 2015, student evaluations reflected that the subject matter was overwhelming understandable, that an atmosphere of mutual respect was established, and that the professor cared about their learning. Students truly appreciated being able to ask questions if they could not make office hours. This presentation will discuss the implementation of the classroom surveys in large versus small classroom sizes in addition to its impact on student learning/evaluations.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Moving a Biochem Majors Lab Course into Discovery Based Research
Authors: Park Chad K.
Published: June 2021

Efforts to establish an upper division lab course based on self‐directed, discovery based biochemical research are detailed. Consideration of the varying student backgrounds and levels of preparation must be balanced with preparation for the departmental requirement of a laboratory based senior thesis. The major goals of this lab course are to provide instruction in scientific writing, as well as in the design, execution, and interpretation of biochemical research projects. The first goal is to train students in reading and understanding original peer‐reviewed scientific reports, as well as in writing in a scientific style (i.e. following Biochemistry format). The second goal is to bridge the gap between receiving detailed lab protocols and the ability to design, implement, troubleshoot and communicate protein based biochemical research investigations in small groups. Our efforts to develop and evolve this course in a land‐grant university are detailed here.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Are Lab Courses Worth the Investment: Factors that Affect Student Success in a Biochemistry Lab Course
Authors: Hart Orla M.
Published: June 2021

There are increasing challenges in maintaining and improving student outcomes in lab courses for a variety of reasons, including the relatively limited time students spend on experimental tasks, to the low credit hour to contact hour ratio. The push to increase enrollment and class sizes means that it is harder for academic programs to prioritize creation of resource‐demanding lab courses. That effective labs must operate in small sections with significant instructor interaction means that there is a disproportionate investment required by faculty, when compared with lecture‐based courses. Course‐based undergraduate research experiences (CUREs) are effective, but may not be suited to service lab courses, or large enrollment lab courses. This study aims to identify factors that directly affect student engagement and success in a service (non‐majors) lab course, which will contribute towards best practices in lab course development and implementation.

Factors investigated in this study were overall course structure, lecture material delivery, lab assignments, general assessment strategies, remediation, and faculty presence during the lab session. The biggest single factor affecting student engagement and learning outcomes was faculty presence in the lab class. When the lab sections were run by TAs without faculty present, the students were more 53% more likely to have negative comments about the course, and did not perform as well (a decrease of almost 10% on final exam scores, and the D, F rate increased from 5.4% to 12.1%). Other significant gains seen in student engagement and learning appear to be as a result of interventions in overall course structure and customized remediation opportunities for students. Assessment in practical skills significantly improved outcomes, without necessarily improving engagement. “Flipping” the lecture delivery from in‐class to online, did not have any impact on engagement or outcomes overall, however there was a significant correlation between when and how long students watched online lecture material, and subsequent exam scores. Interestingly, changing the lab assignments from full lab reports to shorter, more focused assignments, did not impact outcomes, but did appear to increase student engagement.

It is hoped that these results will help validate the impact of undergraduate lab courses, and justify the commitment of resources. Structuring lab courses effectively and efficiently, and assessing lab learning in a way that ensures students receive high quality instruction and feedback, and are achieving the desired outcomes, will mean that the benefits of these courses justify the costs by both faculty and students.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
O‐GlcNAcylase (OGA): A Sugar Cleaver
Authors: Gallen Corey , Rios‐Rosales Anapaula , Jacus Isabella G. , Cobb Allison , Nashman Zarrar , Grant Raheim , Shor Audrey C.
Published: June 2021

A basic component of learning undergraduate biology concepts is grasping the interplay between carbohydrates and proteins. O‐GlcNAcylation is a post‐translational modification that covalently attaches O‐linked N‐acetylglocosamine (O‐GlcNAc) moieties to target proteins. Two enzymes regulate O‐GlcNAcylation; O‐GlcNAc transferase catalyzes the addition of O‐GlcNAc to specific serine and threonine residues, and O‐GlcNAcylase (OGA) hydrolyses the monosaccharide from the substrate. The focus of this project was to design and build a physical model that illustrates the key functional features of human OGA. Two isoforms of the enzyme exist in humans; 1 and 3 which are expressed in the cytoplasm and nucleus, respectively. The cytoplasmic isoform includes a full length protein, while the nuclear isoform is shorter that lacks a C‐terminal acetyltransferase‐like domain and demonstrates reduced enzymatic activity in vitro. Human OGAs possess a fugitive substrate binding groove that is highly conserved through metazoans. Two aspartic acid residues 174 and 175 comprise the catalytic activity of the enzyme and are conserved across all domains. Residues significant to binding O‐GlcNAc are close to the sugar/catalytic machinery or occupy positions shown to undergo a conformational change upon inhibitor binding. Mutations targeting the substrate binding groove identified residues that are not required for hydrolysis, but serve roles in recognition and binding of substrate. The differences between the residues required for the turnover of these substrates may reflect a change in molecular movement in OGA sites. Further elucidation of this mechanism could help advance our treatment and/or prevention of diseases like neurological disorders and diabetes.

Support or Funding Information

Funded in part by NSF‐DUE 1725940 for the CREST Project.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Validation of Arid1a as a Mammary Tumor Driver in Mice
Authors: Winters Marquita , Kartha Nithya , Schimenti John
Published: June 2021

Distinguishing commonly deleted genes as drivers or passengers of human cancers are key to delineating mechanisms of tumor initiation. The “Chaos3” mouse model of spontaneous breast cancer carries a missense mutation in MCM4 (minichromosome maintenance 4), causing destabilization of the MCM2‐7 replicative helicase, which in turn causes elevated genomic instability. Approximately 80% of female mice homozygous for this mutation in the C3H/FeJ background develop mammary tumors with an average latency of 12 months. Genomic analysis of these tumors revealed that ~70% had monoallelic deletion of tumor suppressor Arid1a. Arid1a is a member of the SWI/SNF chromatin remodeling complex and is responsible for conferring specificity of SWI/SNF complexes to their targets. Previous studies in the lab have shown that introduction of a second Arid1a allele in monoallelically deleted, cultured tumor cells rescued the tumorgenic phenotype following transplantation into host mammary glands. We propose that deletion of Arid1a early in postnatal development is one driver of Chaos3 mammary tumors. Here, we will perform intraductal mammary injections of adeno‐cre virus into Chaos3 females bearing a floxed Arid1a allele. Loss of Arid1a has been implicated as a driver of tumorigenesis in many cancers such as clear cell ovarian carcinoma and endometrioid carcinoma, and is deleted in ~5–11% of human breast cancers. Identifying Arid1a as a driver of mammary tumors in Chaos3 mice would validate its potential role in development of sporadic breast cancer in people.

Support or Funding Information

Breast Cancer Coalition of Rochester

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