bluuebunny/crossref_metadata_2025 huggingface dataset:
Our Unique Cell Lines Derived Serially After Injection With Human T24 HRAS Transformed NIH/3T3 Mouse Cells (GhrasT‐NIH/3T3) Showed The Highly Tumorigenic T1‐A Cells Cultured From The First Primary Tumor Had A Six‐Fold Higher LD50 For Resveratrol Killing Than The LD50 For Highly Metastatic Lung T4‐PA Cells Derived After Three Subsequent In Vivo / In Vitro Serial Manipulations
Authors: Ray Durwood Barber , Jones David H.
Published: June 2021

Our new cell lines demonstrating progressively increasingly metastatic behavior as they were derived in a series of in vivo/in vitro manipulations (Figure 1) provide the opportunity to evaluate in vivo and in vitro compounds and methods to kill primary tumor cells and various forms of increasingly metastatic cells derived from the same primary tumor (Figure 2) (Ray, D.B., et. al., Experimental Cell Research, 2016 Jan; 340 (1): pp. 1–11). We determined the dose of the anti‐cancer natural phytoalexin resveratrol to kill 50% (LD50s) of our various cells that represent progressive cancer stages. Cell counts were recorded daily, over the course of 3 to 6 days, using gridded plates and photomicrographs of six pre‐determined 2 mm square sections of each plate. An average percent change in cell number from each individual grid was then calculated. P‐values were determined between each treatment time and dose and significance was determined. The NIH Swiss and NIH/3T3 cells were not significantly affected by 3–300 μM after 3 days. After 3 or 4 days the LD50s for the tumorigenic cells were GhrasT‐NIH/3T3 cells: LD50 = 3.5+/− 1.0 μM; T1‐A cells: LD50 = 45 +/− 5.0 μM; T4‐PA cells: LD50 = 7.0 +/− 1.5 μM. Thus, unexpectantly, the highly tumorigenic T1‐A cells were significantly more difficult to kill than the highly metastatic T4‐PA cells. This pattern may mimic the epithelial to mesenchymal transition (EMT) seen in breast and other cancer progression. EMT enhances mobility, invasion, and resistance to apoptotic stimuli as tumor cells acquire stem cell properties and exhibit marked therapeutic resistance. RNA‐Seq, genome sequencing (WGS) and spectral karyotyping (SKY) analysis of these cells demonstrating increasingly aggressive metastatic cancer tumorigenesis that progressed from a single nuclear genotype and mitochondrial haplotype should help contribute to the understanding of cancer progression. These cells are available by contacting our laboratory.

Support or Funding Information

Supported in part by the National Science Foundation grant # 8602787, the Grove City College Research Fund, the Grove City College Swezey Research Fund and the Grove City College Janicki Foundation Research 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.
Weight loss and fitness intervention increase markers of hepatic bile acid (BA) synthesis, while reducing serum total BA concentrations in sedentary, obese insulin resistant women
Authors: Mercer Kelly E , Pack Lindsay M , Lou Xiangyang , Thyfault John , Campbell Caitlin , Fiehn Oliver , Chandler Carol J , Burnett Dustin J , Souza Elaine C , Casazza Gretchen A , Keim Nancy , Newman John W , Hunter Gary R , Fernandez Jose R , Garvey W Timothy , Harper Mary‐Ellen , Hoppel Charles L , Adams Sean H
Published: June 2021

Beyond adipose and muscle, excerise has profound effects in other metabolically‐important tissues including the liver. Bile acids (BAs) are synthesized from cholesterol via the rate‐limiting enzyme cholesterol 7 alpha‐hydroxylase (CYP7A1) and contribute to the solubilization and absorption of lipid soluble nutrients, and serve as important signaling molecules capable of systemic endocrine function. Circulating BA are known to be higher in obesity and insulin resistance, but effects following exercise and diet‐induced weight loss are unknown. To test if improved fitness and weight loss influence bile acid metabolism and homeostasis, we measured fasting serum concentrations of total BAs (conjugated and free forms of primary and secondary BAs) in sedentary, obese insulin‐resistant women (N=12) before and after a ~14 wk weight loss and exercise intervention [Campell et al. 2014, PLoS One, 9: e84260]. In addition, serum FGF19 (a regulator of BA synthesis) and 7‐alpha‐hydroxy‐cholesten‐3‐one (C4, a verified marker of CYP7A1 enzymatic acitivity) were measured by ELISA and LC‐MS, respectively. Using mixed‐model analyses and the change in relative VO2peak (mL/min/kg) as a covariate, we observed a significant decrease (~30%) in total serum BAs post‐intervention relative to pre‐intervention samples. Corresponding to the decrease in total BAs, fasting serum concentrations of C4 were 55% higher post‐intervention relative to the matched pre‐intervention samples (P=0.004). Interestingly, we observed no changes in serum FGF19. These data suggest that weight loss and/or improved fitness altered BA metabolism marked by increased hepatic BA synthesis and reduced serum BA. The underlying mechanisms remain to be determined, but work from our preclinical rodent models supports the idea that increased BA metabolic flux is linked to enhanced hepatic mitochondrial function.

Support or Funding Information

This work was supported by USDA Projects 6026‐51000‐010‐05S and 2032‐51530‐022‐00D, and NIH‐NIDDK R01DK078328 and NIDDK U24 DK097154.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating Physiological Interactions between the Electrogenic Na/HCO3 Transporter NBCe1‐B and its Cytosolic Binding Partner IRBIT
Authors: Lee Seong‐Ki , Boron Walter F.
Published: June 2021

The electrogenic Na/HCO3 transporter NBCe1 is expressed in numerous organs, where it plays many important roles, including trans‐epithelial HCO3− transport (e.g., pancreatic ducts) and maintenance of intracellular pH (e.g., cells in CNS). Presently, five NBCe1 variants are known: the A variant, mainly in kidney; B, ubiquitous; C, CNS; and D and E, murine reproductive organs. The extreme NH2‐terminus (Nt) of each variant is a strong determinant of transport activity. A and D have a unique Nt of 41 amino acids (aa) that, at least in A, confers high transport activity. B, C, and E have a unique Nt of 85 aa that, at least in B and C, confers low transport activity. However, B interacts with IRBIT [inositol trisphosphate (IP3)‐receptor binding protein released with IP3], which binds to the unique Nt of NBCe1‐B, and markedly enhances the transport activity of NBCe1‐B. Previously, others examined the interaction by fusing a fragment of the unique Nt of NBCe1‐B/C/E to maltose‐binding protein (MBP) for use in a MBP pull‐down assay, and found that NBCe1‐B (e1B) residues 1–62 are necessary for the interaction. In a recent study, we showed that the cationic cluster (residues 40–48 inclusive) is an essential element of the autoinhibitory domain (AID) that confers low activity on e1B. The purpose of the present study is to refine the structural requirements of the IRBIT‐e1B interaction under physiological conditions. Here, we co‐express 7 constructs of e1B (one at a time) together with super‐IRBIT (sIRBIT, which lacks the PP1 binding site) in Xenopus oocytes, followed by two‐electrode voltage‐clamp, extracellular‐surface‐biotinylation, and NeutrAvidin pull‐down of plasma‐membrane proteins. We find that sIRBIT copurifies, as assayed by western blot, with biotinylated e1B protein, but only for low‐activity e1B constructs (i.e., AID intact) for which sIRBIT enhances transport activity. sIRBIT does not copurify with the Δ940–48 e1B construct, which lacks the cationic cluster (i.e., AID disrupted). Thus, the cationic cluster is not only essential for autoinhibition, but also for sIRBIT binding. Our approach will be useful for elucidating other motifs that may be necessary for the interaction of IRBIT with NBCe1‐B, ‐C, or ‐E.

Support or Funding Information

Supported by grants from the Office of Naval Research, N00014‐15‐1‐2060, N00014‐16‐1‐2535

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Varied Effects of Dietary Carotenoid Supplementation on Oxidative Damage in Tissues of Two Waterfowl Species
Authors: Mohr Alex E , Girard Marc , Rowe Melissah , McGraw Kevin J , Sweazea Karen L
Published: June 2021

The antioxidant potential of dietary carotenoids is widely debated for birds, with some studies supporting a protective role whereas others report either no effect or even a pro‐oxidant effect. However, the majority of previous research on this topic has not analyzed the oxidative status of a series of tissues in animals and has not considered a range of carotenoid dosages. Therefore, we investigated the effects of three levels of dietary carotenoid supplementation on plasma, liver, adipose, heart, and breast muscle oxidative damage in two congeneric species of ducks. After a 6‐week depletion period, captive adult northern pintail (Anas acuta) and mallard (A. platyrhynchos) ducks of both sexes were fed either a carotenoid‐depleted diet (<3 μg/g), a carotenoid‐rich diet (50 μg/g) within physiological range, or a carotenoid‐rich diet (100 μg/g xanthophylls) within pharmacological range for 17 weeks. We hypothesized that this range of dietary carotenoid dosages would differently affect oxidative damage, as measured by thiobarbituric acid reactive substances (TBARS), between species and sexes and among the tissues examined. Overall, liver and heart tissue exhibited the highest levels of oxidative damage, whereas adipose tissue had the lowest. We found that dietary xanthophyll (lutein and zeaxanthin) supplementation had no significant effect on tissue pro‐oxidation in males and females from both species. Moreover, for only two tissues (plasma and heart) did we find sex or species differences in oxidative stress. We did not find significant correlations in the levels of oxidative damage among plasma, liver, adipose, heart and breast muscle. In conclusion, the current study does not support a consistent antioxidant role for dietary carotenoids in the tissues of these two waterfowl species. Instead, our results align with the notion that carotenoids play complex, tissue‐specific roles in oxidative balance in birds.

Support or Funding Information

The authors thank the University of Poitiers, France for providing MG with a summer internship to help support this research. The larger study was funded by the National Science Foundation (grant numbers NSF/IOS‐0746364 and ‐0925633 to KJM).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intranasal Administration of MSC‐Derived Exosomes Modulate Status Epilepticus Induced Abnormal Plasticity of Newly Born Neurons and Microglial Activation in the Hippocampus
Authors: Atobiloye Mariam Adebola , Attaluri Sahithi , Gitai Daniel , Kodali Maheedhar , Kim Dong‐Ki , Prockop Darwin J , Shetty Ashok K
Published: June 2021

Status epilepticus (SE) is a self‐sustaining seizure episode that causes adverse alterations in the hippocampus, which lead to a state of chronic epilepsy. We investigated the effects of mesenchymal stem cell (MSC)‐derived exosomes (small extracellular vesicles, EVs) to ameliorate the most prominent early changes induced by SE such as the abnormal plasticity of newly born neurons and activation of microglia. F344 rats were subjected to Kainic‐acid‐induced SE for 2 hours and then intranasally treated with a dose of exosomes (100 billion) or vehicle (VEH). In animals receiving VEH after SE (n=5), 20% of double cortin (DCX)‐positive newly born neurons displayed basal dendrites at 7 days post‐SE. Furthermore, the percentage of microglia with ramified morphology (resting microglia) was severely depleted in the CA1 subfield (6%) of these animals because of their activation. In contrast, in animals receiving EVs after SE (n=8), only 5% of DCX+ newly born neurons exhibited basal dendrites (p<0.0001). Also, these animals displayed a higher percentage (20%) of ramified microglia than animals receiving VEH, implying a reduced level of activation (p< 0.05). Thus, intranasal administration of MSC‐derived exosomes after SE is efficient for dampening the abnormal plasticity of newly born neurons and modulating microglial activation in the hippocampus. Basal dendrites promote abnormal connectivity between dentate granule cells and hilar/CA3 pyramidal neurons, as well as enhance granule cell to granule cell connectivity. Since both abnormal plasticity and neuroinflammation contribute to the occurrence of spontaneous seizures in the chronic phase, the results have significance.

Support or Funding Information

Supported by a DOD grant to A.K.S.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sampling Bias in the Trend of Post‐Pleistocene Gracilization: How Terrain Affects Lower Limb Robusticity
Authors: Zachwieja Alexandra Jeanette , Shackelford Laura
Published: June 2021

Many studies have documented a decrease in modern human lower limb measures of cross‐sectional geometry (CSG) throughout the Pleistocene and Holocene. These decreases are often associated with subsistence changes and technology that allowed for a more sedentary lifestyle (e.g., rise of agriculture). Italian Neolithic samples (19‐4 ka) deviate from this trend, with measures similar to earlier, highly mobile populations (Marchi, 2008; Marchi et al., 2011) despite an overall trend for reduced robusticity through Holocene Europe as a whole (Holt et al., 2018). Active populations with a lifetime of mobility on rough terrain have been shown to have higher cortical thickness and CSG properties that resist bending and torsion in the anterior‐posterior plane – which would not fit within the traditional paradigm. Here, we evaluate changes in femoral and tibial midshaft CSG measures of robusticity. These include maximum and minimum second moments of area (Imax/Imin), second moments of area in the x and y plane (Ix/Iy), polar moment of area (J), and cortical area (CA) for populations in Europe, Africa, and Asia (N = 440) throughout the past 40,000 years (Early Upper Paleolithic, Late Upper Paleolithic, Holocene) using ANOVA and linear interaction models. Generally, Early and Late Upper Paleolithic groups show no significant differences, but Early Upper Paleolithic and Holocene groups were significantly different in all femoral measures, and Late Upper Paleolithic and Holocene groups were significantly different in all measures except Ix/Iy. Linear models showed no significant effect of elevation on CSP or any interaction effects except in femoral CA (Early Upper Paleolithic, p = 0.02; Late Upper Paleolithic, p = 0.03; Holocene, p = 0.01). A paradigm of gracilization throughout time is not supported in Asian populations when they are compared within regions. Reduced robusticity in Holocene Andaman Islanders as compared to Japanese/Vietnamese samples suggest that lower limb robusticity can increase because of activities on mountainous terrain, subverting the traditional paradigm. This suggests that we cannot assume that all populations experienced a reduction in robusticity into the Holocene and we must be aware of the danger of a sampling bias in our work.

Support or Funding Information

This research was supported by Sigma Xi, NSF BCS‐0314002, and the American Museum of Natural History

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Eye‐Tracking Data to Determine what Biochemistry Students Attend to when Completing a Three‐Dimensional Modeling Activity
Authors: Freeman Sienna , Calvert Jenifer , Terrell Cassidy , Randolph Adriane , Cortes Kimberly Linenberger
Published: June 2021

Biochemistry students experience extraneous cognitive load when presented with distracting information while learning a new task. Consequently, the students struggle to create and store connections between old and new concepts. As this phenomenon becomes increasingly common in the classroom, there is a need to identify methods in which instructors can most effectively present new information while ensuring that students both understand the material and retain the new knowledge. In an attempt to solve this issue, the study biochemistry students were asked during a simulated classroom environment to use models of different serine proteases while answering a series of questions exploring the complementarity of the active sites and substrates. Tobii Glasses 2 were used to track student eye movements and fixations while completing the tasks. Using Tobii Pro eye tracking software, we were able to develop heat maps that assist in the identification of areas of interest on the serine protease model and accompanying worksheet during completion of the activity. With these results, we can determine if students are focusing on what the instructor intends during the activity and if not revise the activity to develop more efficient methods for instructors to present the material to their students. This will promote increased retention of the new skills presented and allow for improved processing of information, thus reducing the amount of extraneous cognitive load experienced by the student.

Support or Funding Information

This project is supported by the National Science Foundation under award number IUSE 1711402/1711425 to University of Minnesota, Rochester and Kennesaw State University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Forensic Science as an Engagement Tool for Student Retention and Student Success in General Biology
Authors: Jones Candace
Published: June 2021

Lane College is a historically black college committed to educating underserved minority students. My goal was to give students a diverse teaching and research experience and improve student success in General Biology I by engaging students through forensic sciences. Through the popularity of television shows like The First 48, CSI, NCIS, and Cold Case Files the study of forensic science has flourished and given teachers a method to teach biology, chemistry, and physics. I implemented forensic science in the laboratory in one of my two sections of General Biology I in the Spring 2018 semester. First students studied and determined the items that need to be analyzed from a mock crime scene after being introduced to some basic forensic science techniques in the first week of laboratory. Each additional week the students participated in hair analysis, fiber analysis, soil analysis, synthetic blood tests, chromatography, and DNA fingerprinting. My hope was that in this environment it would foster the students to ask questions, research background information, design experiments, gather and analyze data leading them to understand the method of scientific discovery. Additionally, the students learned to collaborate and build their critical thinking skills while becoming self‐learners. To measure the success of the intervention, I used a mixture of Forensic Science questions (n=25) that was given as a pre and post test to both sections as a way to compare the effect of the intervention. At the end of the course, the class that received the forensic science intervention had a small average increase compared with that of the class did not receive the intervention. When looking at the questions and comparing the pre and post averages 15 out of 25 had an increase 7 out of 25 had a decrease and 3 out of 25 had no change. There was a slight increase in student success in General Biology I by engaging students through forensic sciences.

Support or Funding Information

National Science Foundation Award HRD # 1623340 to MVS

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Peer‐Led Team Learning and Course‐based Undergraduate Research Experiences in General Biology and General Chemistry
Authors: Van Stry Melanie , Gaber Farag , Sklensky Diane , Ude Mba
Published: June 2021

Lane College is a historically black college with a commitment to educating underserved minority students. Our overall goal for this project is to improve student learning of foundational concepts and retention in Biology and Chemistry through student‐centered activities within our first‐year courses. These approaches include implementation of peer‐led team learning strategies and course‐based undergraduate research experiences (CUREs) within the General Biology and General Chemistry courses. Our peer‐mentors, who serve as supplemental instructors for our General Biology and Chemistry courses are an essential element for the success of the program. They receive leadership training, through a collaboration with the Office of the First Year Experience as well as mentored undergraduate research experiences before working with our first‐year students. We have redesigned our laboratory courses to implement CUREs. Biology projects include comparative genomics in collaboration with the Genomics Education Partnership, environmental surveys of antibiotic resistant bacteria or sequence analysis of GAPC from plants growing on campus. Chemistry projects include development of novel analytical methods to measure chemical components of household products, such as using the iodine‐clock reaction to determine the amount of starch in various foods. We are assessing student learning outcomes related to understanding the scientific method using the Experimental Design Ability Test and the CURE survey developed by the GEP. In addition, we are evaluating student learning outcomes of the process of science, such as development and testing of hypotheses, recording observations within a laboratory notebook, and communication of results through oral presentations and written reports.

Support or Funding Information

NSF HRD1623340

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Changing the Ligand Specificity of a Guanine Riboswitch by In Vitro Selection
Authors: Abdullah Hasan P , Morse Daniel Paul
Published: June 2021

Riboswitches are RNA elements in messenger RNAs that regulate gene expression by undergoing a ligand‐triggered conformational change. Riboswitches bind tightly and specifically to their ligands, so they have the potential to serve as highly effective sensors in vitro. We previously modified several purine riboswitches for use in vitro and, indeed, they could detect their ligands with high sensitivity and specificity (1). To extend the utility of our sensor design, we developed an in vitro selection strategy to isolate variants of riboswitches that have altered ligand specificity. We are attempting to find variants of the guanine riboswitch that can detect hypoxanthine rather than guanine. This is a challenging task because hypoxanthine is identical to guanine except for the absence of an exocyclic amino group. We partially randomized the sequence of our guanine sensor, and used hypoxanthine as the ligand during selection. Our first experiment produced only the original, wild‐type guanine riboswitch. In hindsight, this was not surprising since the guanine riboswitch can detect hypoxanthine (albeit poorly), and it was the most abundant RNA in our initial partially‐randomized RNA pool. To solve this problem we designed a new experiment based on the C74U mutant of the guanine riboswitch. C74 forms a Watson‐Crick base pair with the bound guanine ligand. Changing C74 to U results in a riboswitch that cannot bind to guanine or hypoxanthine but can bind weakly to adenine (2). We performed a second selection in which we partially randomized the sequence of the C74U mutant, except every variant retained a U at position 74. Since the C74U mutant cannot bind to hypoxanthine, we reasoned that we could select variants that could do so by allowing hypoxanthine to form a “wobble” pair with U74. We also anticipated that we could isolate hypoxanthine sensors that discriminate against guanine due to steric clashes between the sensor and the exocyclic amino group of guanine. After 11 rounds of selection, the selected RNA pool could no longer detect adenine but could detect hypoxanthine and guanine. The sensitivity for hypoxanthine and guanine was much lower than that of the wild‐type guanine riboswitch. However, the selectivity for hypoxanthine versus guanine was slightly improved, suggesting that the pool may contain individual variants that can discriminate against guanine. We are currently determining the sequence of a few variants in the selected pool to identify RNAs that we would like to test individually for their ability to specifically detect hypoxanthine.

Support or Funding Information

Defense Threat Reduction Agency [MIPR HDTRA1620511 to D.M.] Office of Naval Research Chemistry Department of the U.S. Naval Academy

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Studies on the HERV‐K Nuclear Export RNA Element
Authors: McShane Erin , Fan Lixin , Kondrup Benjamin , Wang Yun‐Xing , O'Carroll Ina
Published: June 2021

Human endogenous retroviruses (HERVs) are remnants of ancient germline infections that comprise 8% of the human genome. A number of HERVs are actively expressed—their products assume important physiological roles in embryonic development and have been associated with cancer and autoimmune diseases. Expression of a type of HERV, HERV‐K, has been associated with amyotrophic lateral sclerosis (ALS) and some cancers. Expression of the Env protein appears to be dependent on a cis‐acting signal, RcRe, and a viral protein called Rec that mediate nuclear export of unspliced mRNAs in Crm1‐dependent manner, similar to the Rev‐RRE system in HIV. Despite low sequence similarity, the HERV‐K RcRE and HIV RRE are both characterized by a long stem and several smaller stems centered around a junction at the top of the long stem. We recently reported the three‐dimensional structure of the HIV RRE RNA and found that it is a flat, “A”‐shaped molecule (Fang et al., 2013). Based on apparent secondary structure similarities, we hypothesized that the unique “A” shape may be conserved in complex retroviruses. We examined the tertiary structure of the RcRE RNA in solution using small‐angle X‐ray scattering. Our results suggest that the two divergent retroviral elements are structurally conserved, attesting to the significance of the three‐dimensional structure in Rev‐RRE recognition. We report a structure‐ and function‐based mutational analysis of the RcRE tertirary structure.

Support or Funding Information

Naval Academy Research Council (NARC)

DTRA CB Technologies Service Academy Research Initiative.

US Naval Academy, Chemistry Department

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of Substrate Length on Lysine Deacetylase Activity
Authors: Nichols Kyara A , Phillips Tiffany , Toro Tasha B , Watt Terry J
Published: June 2021

Lysine deacetylases (KDACs) are proteins involved in regulating post‐translational modification of lysine residues in proteins. Anomalous acetylation of lysine has been linked to numerous diseases, and unusual activity of lysine deacetylases often appears to be a causative factor. Our goal is to understand the substrate specificity of metal‐dependent KDACs. Here we are specifically interested in the correlation between KDAC activity with peptides derived from acetylated proteins and the activity of the same KDAC with the corresponding full‐length substrate proteins. Activity with peptides is determined by both label‐free fluorescence assays and mass spectrometry. Initial results with peptides, each derived from a single source protein but of varying length of sequence on either side of the acetylated lysine, did not reveal a correlation between peptide length and KDAC activity. The absence of such a correlation suggests important contributions from secondary structure of the substrate protein and/or contributions from amino acid residues distant from the acetylated lysine in sequence. Ongoing work with full‐length substrate proteins, obtained through recombinant expression with a site‐specific acetyllysine or using immunoprecipitation from human cell lines, provides further insight into substrate features contributing to KDAC activity. Correlation, or the lack thereof, of the activity of the KDACs with the peptide models and full‐length substrates serves as an important control on models predicting KDAC activity, which are typically based on peptides of limited variability. Understanding how substrate size and structure affect the overall activity of KDACs will also provide insight into their substrate specificity and biological targets.

Support or Funding Information

The Louisiana Cancer Research Consortium, National Institutes of Health 5G12MD007595 and TL4GM118968, National Science Foundation CHE 1625993 and MCB 1817358, and the U. S. Army Research Laboratory and the U. S. Army Research Office W911NF1810450.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Domain Specific Substrates of Lysine Deacetylase 6
Authors: Parham Imari , Eyassu Daniel G , Toro Tasha B , Watt Terry J
Published: June 2021

Lysine deacetylases (KDACs) are enzymes that regulate the post‐translational modification of lysine by acetate. KDACs serve an important role in numerous biological processes as well as diseases such as asthma, diabetes, and certain cancers. Unlike other metal‐dependent KDACs, KDAC6 contains two catalytic domains. We hypothesize that the catalytic domains of KDAC6 catalyze deacetylation reactions independently, with each domain having several distinct substrates. Several putative substrates have been linked to KDAC6, but which catalytic domain is relevant for deacetylation is generally unclear. We have recombinantly expressed the full‐length KDAC6 as well as the individual catalytic domains. Activity of each construct was measured with peptides derived from the putative substrates, using a combination of a label‐free fluorescence assay and mass spectrometry. Activity was also determined with a selection of full‐length substrate proteins. Differences in activity of the two domains, as well as compared to the full‐length KDAC6, provide insight into the substrate specificity and relative activity of each domain. Computational methods using recently reported crystal structures of the KDAC6 domains and the tested substrates provides additional information about substrate interactions. The identification of independent domain activity and domain specific substrates using these techniques will provide the basis for a greater understanding of the biological roles of KDAC6.

Support or Funding Information

The Louisiana Cancer Research Consortium, National Institutes of Health 5G12MD007595, TL4GM118968, UL1GM118967, and R15GM129682, and National Science Foundation MCB 1817358.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analyzing Protein Kinase C Interactions With Rho‐type GTPases in the Filamentous Fungus Aspergillus nidulans
Authors: Carroll Maleiah , Craft Alexis , Freyaldenhoven W. Toler , Betton Brianna , Campbell Lynsey , Hameed Muhammad Y. , Vanderwall David , Hill Terry , Jackson‐Hayes Loretta
Published: June 2021

Cell division in filamentous fungi involves coalescence and subsequent constriction of a contractile actomyosin ring (CAR), as in other fungi and metazoans. In fungi, constriction of the CAR is followed by deposition of cell wall material resulting in a septum. In the model organism, Aspergillus nidulans, many proteins are involved in septation including protein kinase C (PkcA), the formin SepA, and the GTPase Rho4. The current study focuses on the relationships among PkcA, SepA, and Rho‐type GTPases during septation. PkcA contains two putative N‐terminal Rho binding domains designated HR1A and HR1B. The formin SepA belongs to the Diaphanous‐related family of formins that contain an N‐terminal Rho binding domain that upon binding of the appropriate Rho GTPase results in formin activation. Reports from another group suggest that in A. nidulans Rho4 is likely the GTPase required for SepA activation. In previous work we showed that PkcA both physically and functionally interacts with SepA via one of its three formin homology domains (FH2), and in the sepA1 temperature sensitive mutant (FH2 domain‐L1369S) which is aseptate at elevated temperature, PkcA fails to localize to septation sites at elevated temperature. Here we report via yeast two‐hybrid assay that PkcA interacts with SepA via one of its putative Rho binding domains (HR1A), and that expressing extra copies of pkcA in a rho4‐null mutant increased colony diameter and improved sporulation. PkcA localization to septation sites is reduced in a rhoA mutant while septation is unaffected. The spatiotemporal relationship among PkcA, SepA, Rho4, and RhoA will be further investigated via yeast two‐hybrid, bimolecular complementation assays, and localization of fluorescence labeled proteins in mutant strains.

Support or Funding Information

Support for this work was provided by the National Science Foundation Grant 1615192 (Loretta Jackson‐Hayes, PI).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Studying The Effect of O‐GlcNAc Modification and Other Stabilizers on Nod1 and Nod2
Authors: Drake Walter Richard , Hou Ching‐Wen , Ukaegbu Ophelia , Zachara Natasha , Grimes Catherine Leimkuhler
Published: June 2021

In order to distinguish infectious pathogens from the trillions of commensal bacteria on the body, humans utilize their innate immune systems, which deploy pattern recognition receptors (PRRs) to detect microbe associated molecular patterns (MAMPs) specific to certain microorganisms. The PRRs Nod1 and Nod2 detect MAMPs found in the peptidoglycan (PG) of bacterial cell walls, and upon binding initiate an immune response via the NF‐kB and other pathways, leading to the expression of pro‐inflammatory cytokines and chemokines. Misregulation of Nod1 signaling is associated with various diseases, including gastric ulcers, stomach cancer, and lung cancer. Improper Nod2 signaling has been linked to Crohn's disease, and three different Crohn's associated variants of Nod2 lead to increased susceptibility to the disease and are unstable relative to the wild type. The enzyme O‐GlcNAc Transferase (OGT), which adds a GlcNAc monosaccharide to certain serines/threonines, post‐translationally modifies both Nod1 and Nod2. This post‐translational modification (PTM) is common among thousands of proteins in the nucleus, membrane, and cytosol, and is mediated by only two enzymes: OGT, which adds GlcNAc, and O‐GlcNAcase (OGA), which removes it. The effect of this PTM is diverse among the numerous client proteins. Targets include transcription factors, including oncogenes and tumor suppressors, as well as insulin receptors, kinases, chaperones, and others. We investigate the role of O‐GlcNAcylation on Nod1 and Nod2. Modification increases the stability of both PRRs according to multiple assays, including a cellular thermal shift assay (CETSA) that measures thermal stability and a cycloheximide‐chase assay that measures cellular proteosomal degradation rates. O‐GlcNAc modification also leads to increased PG‐induced NF‐kB activity. The Crohn's associated variants of Nod2 are modified by OGT as well, and the modification rescues the particularly unstable 1007fs variant to near‐wild type. Additionally, we measure the stability effects gained from PG fragment binding and chaperone proteins.

Support or Funding Information

MITZUTANI GLYCOSCIENCE FOUNDATION

NATIONAL SCIENCE FOUNDATION (CAREER CHE 1554967)

NIH NATIONAL HEART, LUNG, AND BLOOD INSTITUTE (P01HL107153 to NEZ)

NIH NIGMS COBRE P20GM104316‐01A1

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase (RibB) of riboflavin biosynthesis has a mononuclear magnesium active site
Authors: Kenjic Nikola , Moran Graham R , Lamb Audrey L
Published: June 2021

Riboflavin (vitamin B2) is a precursor for the synthesis of the cofactors FAD and FMN. Like other vitamins, riboflavin cannot be synthesized by mammals and must be obtained from the diet. Bacteria and plants synthesize riboflavin in five steps using the enzymes RibA‐E. The focus of this study is the 3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase, otherwise known as RibB. RibB is a magnesium dependent enzyme that catalyzes the conversion of the sugar ribulose‐5‐phosphate (Ru5P), a product of the pentose phosphate pathway, into 3,4‐dihydroxy‐2‐butanone‐4‐phosphate (DHBP). The reaction catalyzed by RibB is an unusual deformylation reaction in which the fourth carbon of the five carbon sugar is removed as formate. Bacher and colleagues1 have proposed that the mechanism is dependent on a skeletal rearrangement linking C3 and C5. We propose a fragmentation mechanism, in which the bond between carbons C3 and C4 is broken allowing formation of the new link between carbons C3 and C5 followed by release of formate. To test these competing hypotheses, we are using NMR and X‐ray crystallography to observe the intermediates formed during catalysis. We have produced 13C labeled Ru5P for use in NMR experiments and shown enzymatic formation of DHBP and formate. We also show the 1.1 Å structure of apo RibB. Previously reported product bound structures of RibB have been modeled with two metal ions in the active site2. However, we report here intrinsic tyrosine fluorescence and activity assay profiles that indicate that RibB requires a single magnesium for full activity.

Support or Funding Information

Weaver Graduate Student Fellowship, the National Science Foundation CHE‐1403293, The University of Kansas 2018 General Research 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.
Salt dependence of a self‐splicing enzyme from an extreme halophile
Authors: Cawood Alexandra , Mills Kenneth V
Published: June 2021

Protein splicing is a post‐translational process facilitated by an intervening polypeptide called an internal protein, or intein. The intein is located between two flanking polypeptides, called exteins. The intein catalyzes its excision from the extein concomitantly with the ligation of the exteins. The extremophile Haloferax volcanii exists in a high salt environment. We are interested in the halophile because we want to learn how efficiently the intein that has invaded its PolB gene splices as a function of salt concentration. We have shown that we can induce splicing of the intein in vitro as a function of salt concentration and time. Others (http://www.ncbi.nlm.nih.gov/pubmed/27462108) have demonstrated that the intein has an active homing endonuclease domain in vivo, and that the presence of the intein has a high fitness cost for the organism. We are interested in studying the endonuclease activity of the intein in vitro, given that a salt‐dependent conditionally‐active nuclease may have utility in biotechnology applications.

Support or Funding Information

This work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Under Pressure: Pressure and Temperature Dependent Nuclease Activity
Authors: Wibben Amanda C , Comeau Hannah Y , Mills Kenneth V
Published: June 2021

An intein is an intervening protein segment that can be found in proteins from extremophiles and is typically self‐excised to allow for complete flanking protein activity. We are interested in studying specific inteins that contain a homing endonuclease (HE), an enzyme that cleaves double stranded DNA in order to initiate insertion of an intein gene by homologous recombination. We are focused on the homing endonuclease activity of inteins from two hyperthermophilic archaea: Thermococcus barophilus (Tba), isolated from a deep sea hydrothermal vent, and Thermococcus kodakarensis (Tko), isolated from a volcanic area on the shore of a Japanese island. Because these intein‐containing extremophiles have been isolated from very high temperatures but from different depths, we are interested in the differential homing endonuclease activity of these extremophiles at varied temperatures and pressures. We have shown that Tba HE is active at pressures up to 1000 atm. Given that Tko is a surfaced‐welling organism, we suspect that the Tko HE will be pressure sensitive while Tba, which lives naturally at a pressure near 400 atm, is not. Additionally, we have found that both Tba HE and Tko HE show temperature dependence, which we attribute to the extreme temperatures that both organisms reside in.

Support or Funding Information

This work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Analysis of Halophilic and Thermophilic Inteins
Authors: Conroy Owen Ryan , Chavez Angel , Mills Kenneth Vincent
Published: June 2021

Protein splicing is a post‐translational modification by which a segment of protein, known as an intein, self‐catalyzes its excision from the flanking polypeptides while simultaneously ligating the exteins. Inteins can be found in many types of archaebacteria, including halophiles, which thrive in environments with high salt concentrations. Two halophilic inteins that interrupt the Cdc21 gene, CdcA and CdcD, have been shown to splice at different salt concentrations. In addition, the PolII intein from the extreme thermophile Pyrococcus horikoshii requires incubation at high temperature in order to splice. We are interested in solving the structure of all three inteins by NMR to better probe the relationship between a protein's structure and its requirements of extreme conditions for activity. To obtain the proteins at sufficiently high concentrations for structural analysis, we sub‐cloned the inteins of interest into expression vectors with an N‐terminal GB1 tag and optimized expression conditions.

Support or Funding Information

This work was supported by the National Science Foundation (MCB‐1517138, KVM) and the Dreyfus Foundation (KVM).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enzymology of Columbamide Biosynthesis
Authors: D'Agostino Gabriel , Manley Olivia M. , Makris Thomas M.
Published: June 2021

Marine cyanobacteria represent a unique, underexplored reservoir of bioactive natural products. Recent genomic analysis and mass spectrometry studies have revealed a novel chlorinated compound known as columbamide from Moorea bouillonii PNG isolated off the coast of New Guinea. Mass spectral shows columbamide to be a dihalogenated compound with chlorine atoms appended to the C6 and C12 positions of a hydrocarbon chain that is thought to be constructed from dodecanoic acid. Genomic studies have proposed a pathway for columbamide biosynthesis but it has yet to be supported by in vitro studies. Of particular interest, the enzymes proposed to catalyze the halogenation reactions, ColD and ColE, are most homologous to dinuclear iron‐dependent enzymes. Although diiron enzymes have been shown to have a great deal of functional diversity, halogenation is not amongst these reported activities. Our study seeks to characterize and reconstruct the early stages of the biosynthetic pathway by generating protein‐tethered substrates for the halogenases ColD and ColE. ColC is believed to be an acyl‐carrier protein for dodecanoic acid as well as a substrate of ColD/ColE. The first enzyme encoded by the gene cluster, ColA, is believed to attach dodecanoic acid to ColC. To investigate the biosynthetic pathway leading to halogenation, the role of ColA will be confirmed, and its enzymology characterized. Additionally, the well‐characterized enzyme acyl‐acyl carrier protein synthetase from the organism Vibrio harveyi will be used to efficiently generate large amounts of acylated‐ColC with non‐native fatty acids. Our study will seek to use the generated substrate‐library in concert with various spectroscopic techniques—such as optical spectra analysis, rapid‐mix stopped‐flow kinetics, and electron paramagnetic resonance (EPR)—to characterize and elucidate the mechanistic details of enzymes ColD/ColE.

Support or Funding Information

National Science Foundation; South Carolina Honors College‐‐Undergraduate Research Funding

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Fitness Cost of Two Inteins in Halobacterium salinarum”
Authors: Exconde Patrick M. , Janton Christopher J. , Makkay Andrea M. , Papke R. Thane , Gomez Alvin V. , Mills Kenneth V.
Published: June 2021

Halobacterium salinarum is from the domain Archaea and an obligate inhabitant of extremely saline conditions, including saturated brines. Its PolII and Cdc21a genes are both interrupted by an intein. Inteins are genetic elements within an organism's genome, which, at the protein level, self‐catalyze their own excision to concomitantly form a functional protein. We previously showed that protein splicing of these inteins depends on high salt concentrations, requiring a minimum of 2.0 M NaCl to be active. This suggests that the intein may be active in the native cellular environment of H. salinarum, allowing for production of functional protein, and that its presence might not be detrimental to its host. Conversely, a study by Naor et al. (PNAS (2016) 113(32), E4654) showed that in another halophilic archaeon, Haloferax volcanii, there is a > 7% fitness cost for maintaining the polB‐c intein. We are interested in whether the inteins in H. salinarum exert a similar fitness cost, and will test this using a growth‐competition assay between wild type and intein‐less strains of H. salinarum.

Support or Funding Information

This work was supported by NASA (grant NNX15AM09G to RTP), the National Science Foundation (grants MCB‐1517138 to KVM) and the Dreyfus Foundation (KVM).

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