bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determinants of Lysine Deacetylase 8 Substrate Specificity
Authors: Toro Tasha B , Swanier Jordan S , Watt Terry J 
Published: April 2020

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. Lysine deacetylases (HDACs or KDACs) are enzymes that reverse this post‐translational modification. Deacetylation is important for many cellular processes and aberrant KDAC activity has been linked to numerous diseases. While proper deacetylation is known to be critical for proper cellular function, details regarding substrates of particular KDACs and how substrate specificity is determined are lacking. To investigate how particular KDACs differentiate between potential substrates, we constructed a structural alignment of several KDAC catalytic domains. We observed that a loop important for substrate binding always contains an aspartic acid residue, but that the position of this residue differs substantially between KDAC8 and other KDACs. Furthermore, molecular dynamics simulations predict that for several peptide substrates which KDAC8 can deacetylate, ionic interactions likely occur between the aspartic acid in KDAC8 and positively charged residues adjacent to the acetylated lysine in substrates, but likely not with other KDACs. In vitro assays with purified, recombinant KDACs support these predictions. For KDAC8, but not other KDACs, a positively charged residue in the −1 position relative to the acetylated lysine significantly influences the enzyme activity with the peptide. In addition, KDAC8 variants lacking the aspartic acid exhibit greatly reduced activity with the same substrates, consistent with our model that this charged residue is important for KDAC8 substrate selection. However, the substrate discrimination within this set of peptides cannot be entirely explained by the aspartic acid residue, so we are investigating other neighboring residues in silico and in vitro for potential contributions. To complement these studies, we are also utilizing a cell‐based approach to identify novel biological substrates, which can be used to further test our hypothesis that there are critical ionic interactions allowing KDAC8 to bind its preferred substrates. In total, these experiments give us a better understanding of how particular KDACs discriminate between the thousands of potential substrates present in cells, as well as information about biological substrates for KDAC8.Support or Funding InformationNIH TL4GM118968, UL1GM118967, 5G12MD007595, and R15GM129682; NSF CHE 1625993 and MCB 1817358; U. S. Army Research Laboratory and the U. S. Army Research Office W911NF1810450; and the Louisiana Cancer Research Consortium.
Mode of Ossification and Extracellular Fiber Characterization of Osteoderm Matrix in the American Alligator (Alligator mississippiensis) and Comparisons to Inherited and Acquired Heterotopic Ossification Disorders
Authors: Dubansky Brooke , Raney Jacob , Dubansky Benjamin 
Published: April 2020

Osteoderms are bones that form in the dermis of several reptilian species and have been a source of scientific curiosity since at least the 1800s. Though there are several hypotheses for their function, a consensus has not yet emerged. The function of osteoderms is further obscured, since there is little histomorphological information on these structures. Indeed, understanding the cellular and molecular events that initiate and drive osteoderm development may clarify the functional significance of these structures. Dubansky & Dubansky (2018) conducted the first histological description of the development of the osteoderm in the American alligator, and identified discrete developmental stages based on the organization of collagen fibers and contributing cell mediators, as well as the degree of mineralization of the matrix. However, questions remain regarding the type of collagen and the identity of the cell precursors that are involved at different stages, as well as the existence of a hyaline cartilage model. In this study, differential staining techniques characterized changes in the fibrous component of the developing matrix of the osteoderm, and investigated whether ossification proceeds via an endochondral mechanism. Early stage osteoderms exhibit an extensive reticulin (type III collagen) fiber scaffold, which is replaced by disorganized, thick type I collagen fibers in mineralizing maturing matrix, and highly organized, thin type I collagen at the proliferating edge of the matrix. Despite morphological similarities to cartilage in some areas, staining showed that hyaline cartilage was absent and, therefore, the mode of ossification more resembled intramembranous ossification, than endochondral ossification. Osteoderm development resembles Heterotopic Ossification (HO) disorders in humans and is being used as a model to study several forms of this pathology. Based on this study, osteoderm histomorphology most closely resembles the inherited disorder Progressive Osseous Heteroplasia, in which pathological bone formation is restricted to the dermis and superficial fascia and proceeds via intramembranous ossification. Documenting similarities and differences among the various types of HO lesions and osteoderms may provide insights into the disease progression and future therapeutic strategies.Support or Funding InformationThis research was supported by a Faculty‐Student Research and Creative Activity Internal Grant from Tarleton State University, Office of Research and Innovation.
Characterization of Islet Health and Function Prior to Transplantation
Authors: Weber Craig , Hart Nathaniel , Steyn Leah V. , Papas Klearchos , Lynch Ronald M. 
Published: April 2020

Transplantation of Islets of Langerhans offers a cell‐based insulin replacement therapy for Type 1 diabetes but requires lifelong immunosuppression. Devices that encapsulate transplanted islets or insulin‐producing cells can isolate and protect islets from the immune system eliminating need for immunosuppression. However, the required cell density may promote a hypoxic environment with subsequent beta cell dysfunction. We investigated the effects of a hypoxic environment on unencapsulated islet insulin secretion by incubating isolated human islets at low (1%) and normal (21%) oxygen tensions. After 24 hrs, the basal secretion rate (BSR) for islets maintained at 1% O2 (4.5 +/− 0.34 pg insulin/20min*ng DNA) was elevated compared to islets maintained in normoxic conditions (21%, 2.0 +/− 0.7 pg insulin/20min*ng DNA). For islets exposed to normoxia the stimulation index (SI) was 6.4 +/− 1.3. The SI in islets exposed to hypoxia was reduced by 50% (3.2 +/− 1.1). Thus, sufficient islet oxygenation is important for human islet function.To investigate the effect of hypoxic conditions on macroencapsulated islet function we placed a high density of rat islets, without supplemental oxygen, into a macroencapsulation device. By perifusion, we observed a delayed and decreased 1st phase insulin secretion when compared to free islets. After 4 days of high density culture within the macroencapsulation device, macroencapsulated rat islets assayed in static assays, showed increased basal secretion, reduced GSIS, and decreased insulin content when compared to free islets.To evaluate free and macroencapsulated cellular function we recently developed perifusion chambers capable of simultaneous measurement of oxygen consumption rate (OCR) and hormone secretion. We found that GSIS was correlated with increased OCR in substrate free media demonstrating the possible utility of dynamic OCR measurements as a real time assay for islet viability and potency. Islet function is sensitive to oxygen tension and metabolic exchange, and using new technologies we are developing strategies to characterize these effects to optimize cellular health and function.Support or Funding InformationJuvenile Diabetes Research Foundation Award #2‐SRA‐2018‐685‐S‐B. NIH Diabetes Impact Award #1DP3DK106933‐01
Combined Effects of Lunar Gravity and Heavy Ion Exposure on Skeletal Muscle Anabolic Signaling
Authors: O’Reilly Colleen L. , Wiggs Michael , Hogan Harry A. , Bloomfield Susan A. , Fluckey James 
Published: April 2020

Unloading‐induced atrophy and exposure to space radiation are considered major physiological limitations to future space exploration. Muscle mass and muscle strength loss have been reported in models of hindlimb unloading and have been associated with fiber type changes and a shift toward more glycolytic activity. It is commonly purported that skeletal muscle is radio‐resistant due to its post mitotic state, but recent work has indicated changes in both protein synthesis rates and muscle morphometry with exposure to high‐charge high energy particles. While unloading‐induced atrophy has been widely studied in models of complete unloading, information on muscle atrophy in partial loading conditions is much less common, but there is indication that muscle anabolism is linked to gravitational load or lack thereof. It is clear that space exploration includes both space radiation‐ and unloading‐induced atrophy; however, the combined effects of these two environmental factors has limited investigation. The purpose of this study was to determine the impact of acute radiation exposure, with or without the addition of partial loading on skeletal muscle anabolic signaling.METHODSBALB/cByJ (4mo female) mice were assigned either to full weight bearing or partial weight bearing equal to 1/6th bodyweight groups. These groups were then further divided into either single 0.5 Gy of 300 MeV whole–body 28 Si radiation exposure (GCR) or sham. Radiation exposure occurred on Day 0 at Brookhaven National Laboratory and was followed by 21d of Earth (1G) or Lunar (G/6) loading. Muscles of the hind limb were examined for common markers of protein anabolism. Protein expression was measured in the quadriceps using western blot techniques and fiber type was measured using immunohistochemistry.RESULTSOur data indicate that neither GCR exposure nor Lunar loading alone altered mTORC1 dependent signaling. DEPTOR expression was significantly lower when lunar loading and radiation were combined, which may be an indication of bolstered repair response (P<0.05). Interestingly, radiation exposure had higher phosphorylation of ERK1/2 when compared to SHAM counterparts (CC 3.2 fold increase, and 5.6 fold increase in G/6 groups), and was associated with the preservation of muscle mass and the predominance of type 2 fiber composition in the irradiated animals of this experiment.CONCLUSIONThis study suggests that heavy ion exposure affects skeletal muscle and can lead to discrete and combined effects in anabolic signaling.Support or Funding InformationThis study was funded in part by National Space Biomedical Research Institute.
The Influence of Age at Natural Menopause on Cerebrovascular Reactivity
Authors: Senese Katherine A. , Corkery Adam T. , Howery Anna J. , Pearson Andrew G. , Miller Kathleen B. , Eisenmann Nicole A. , Barnes Jill N. 
Published: April 2020

The onset of menopause coincides with a loss of the vasoprotective effects of estrogen. Early menopause is associated with increased cardiovascular risk factors and cardiovascular disease; however, the specific cerebrovascular outcomes regarding the timing at natural menopause are not understood. The purpose of this study was to investigate how the age of natural menopause influences cerebrovascular reactivity, a measure of cerebrovascular function. We studied 37 women with earlier (Early; n = 20; age at onset of menopause = 46 ± 3 y; age at time of study = 61 ± 4 y) and later (Late; n = 17; age at onset of menopause = 55 ± 2 y; age at time of study = 62 ± 3 y) onset of natural menopause. Supine brachial blood pressure was taken in triplicate using a sphygmomanometer before study procedures. Middle cerebral artery velocity (MCAv), mean arterial blood pressure (MAP), and end‐tidal carbon dioxide (ETCO□) were continuously recorded during hypercapnia to measure cerebrovascular reactivity. Cerebrovascular conductance index (CVCi) was calculated as MCAv/MAP. Reactivity was calculated as the slope of the relationship between ETCO□ and each variable of interest. There were no significant differences in MCAv reactivity (Early: 2.0 ± 0.2 cm/s/mmHg; Late: 2.3 ± 0.2 cm/s/mmHg; p = 0.09), MAP reactivity (Early: 1.0 ± 0.3 mmHg/mmHg; Late: 1.0 ± 0.2 mmHg/mmHg; p = 0.50), or CVCi reactivity (Early: 0.01 ± 0.002 cm/s/mmHg2; Late: 0.02 ± 0.001 cm/s/mmHg2; p = 0.16) between women who experienced an earlier or later onset of menopause. However, the Early and Late groups had a statistically significant difference in supine brachial MAP at rest (Early: 99.5 ± 1.9 mmHg, Late: 91.6 ± 2.9 mmHg; p < 0.05), which may confound cerebrovascular reactivity to hypercapnia. When brachial MAP was added as a covariate, the Early group had a significantly lower MCAv reactivity (p < 0.05) than the Late group. These results suggest that the age at natural menopause may influence cerebrovascular function in postmenopausal women, as women who experience an earlier onset of menopause have lower cerebrovascular reactivity after resting MAP is accounted for. Future studies should examine intervention strategies, such as menopausal hormone therapy, in order to mitigate these discrepancies in cerebral blood flow regulation.Support or Funding InformationResearch funded through a grant from the Alzheimer’s Association.
Exploring a Role for Organic Cation Transporter 3 in Ethanol and Cocaine Co‐Abuse
Authors: Bowman Melodi Ann , Owens W. Anthony , Vitela Melissa , Koek Wouter , Daws Lynette C. 
Published: April 2020

According to the National Institute on Drug Abuse, concurrent use of cocaine and alcohol is a major cause for emergency hospitalization, underscoring a vital need to understand the mechanistic basis of this highly addictive, and dangerous drug combination. It is well‐known that both cocaine (blocker of dopamine, norepinephrine and serotonin transporters, DAT, NET and SERT, respectively) and ethanol (EtOH) increase extracellular dopamine (DA), norepinephrine and serotonin. However, we and others, find no evidence for EtOH interacting with DAT, NET or SERT, suggesting that EtOH may be acting elsewhere to inhibit uptake of these monoamines. Organic cation transporter 3 (OCT3) is emerging as an important player in regulation of monoamine signaling. However, whether OCT3 contributes to actions of abused drugs that act primarily by increasing extracellular levels of monoamines, remains unclear. To establish proof‐of‐principle for this idea, we conducted studies to determine the ability of EtOH to inhibit uptake of the prototypical cation [3H]1‐methyl‐4‐phenylpyridinium ([3H]MPP+) into cultured HEK293 cells stably overexpressing human OCT3, and found the IC50 to be 4 mM. Extracellular brain concentrations of EtOH reported after doses that produce effects ranging from mild sedation to full anesthesia in rodents range from 0.5–35 mM. Thus, these data provide support for OCT3‐dependent actions of EtOH, and show that concentrations of EtOH reaching brain following behaviorally relevant doses may be sufficient to engage OCT3. We also found that cocaine does not have activity at OCT3 raising the possibility that EtOH may interact with OCT3 to inhibit uptake of monoamines, potentially increasing the addictive properties of cocaine and propagating the concurrent use of these drugs. To this end we used high‐speed chronoamperometry to interrogate the effects of local application of EtOH, cocaine, and their combination on clearance of exogenously applied DA from extracellular fluid in striatum of male and female wild‐type mice in vivo. We found that, contingent upon concentration of DA applied, EtOH enhanced the ability of cocaine to inhibit DA clearance (low and high DA), and prolonged the duration that cocaine inhibited DA clearance (high DA). This effect trended to be greater in female mice. Initial studies in males show that the ability of EtOH to inhibit DA clearance is lost in OCT3 KO mice, as is EtOH‐enhancement of cocaine‐induced inhibition of DA clearance. Studies investigating the effect of EtOH on conditioned place preference for cocaine, and cocaine‐induced locomotion are ongoing. Taken together, results so far suggest that potentiation of the neurochemical and behavioral effects of cocaine by EtOH are OCT3 dependent. OCT3 may be a putative target for therapeutic intervention in the treatment of EtOH and cocaine co‐abuse.Support or Funding InformationSupported by R21 DA046044 to LCD and WK
Fam83d is Induced During Neurogenic Skeletal Muscle Atrophy and Modulates MAP Kinase and AKT Signaling
Authors: Waddell David , Cooper Lisa , Kavanagh Jack 
Published: April 2020

Skeletal muscle atrophy is a potentially debilitating condition resulting in muscle weakness and a decrease in muscle mass in response to a wide range of physiological stimuli including aging, cancer, denervation, and corticosteroid exposure. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated mouse gastrocnemius muscle from mice at 3 and 14 days following sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Fam83d is expressed in skeletal muscle and is significantly induced in response to denervation. Quantitative PCR (qPCR) and Western blot analysis revealed that Fam83d is more highly expressed in proliferating myoblasts compared to differentiated myotubes. Ectopic expression of myc‐tagged Fam83d revealed that the protein is rapidly turned over in a 26S proteasome‐dependent manner as myoblasts begin to differentiate. Characterization of the transcriptional regulation of Fam83d was analyzed by fusing the proximal promoter region to a reporter gene. The proximal promoter region contains putative E‐box consensus elements that facilitate binding of myogenic regulatory factors (MRFs) and transfection of the reporter construct in combination with MyoD inhibited Fam83d reporter gene activity. To assess subcellular localization, Fam83d was fused with green fluorescent protein (GFP) and the fusion protein was expressed in C2C12 mouse myoblasts cells, visualized by confocal microscopy, and observed to localize exclusively to the cytoplasm. In addition, Fam83d was ectopically expressed in cultured muscle cells and markers of muscle cell differentiation, MAP Kinase signaling, and AKT/mTOR signaling were analyzed. Fam83d overexpression resulted in significantly repressed levels of myosin heavy chain and myogenin expression, while levels of phosphorylated ERK, phosphorylated AKT and total AKT were dramatically repressed. Interestingly, disruption of a putative phospholipase D‐like (PLD‐like) domain significantly attenuated the Fam83d‐mediated repression of both MAPK and AKT/mTOR signaling. Interestingly, it was also observed that Fam83d physically interacts with and destabilizes CKIα in a PLD‐like domain‐dependent manner. The discovery that Fam83d is expressed in skeletal muscle combined with the observation that Fam83d, a potential modulator of MAP Kinase and AKT signaling, is induced in response to neurogenic atrophy helps further our understanding of the molecular and cellular events of skeletal muscle wasting.Support or Funding InformationThe work described in this study was supported by UNF Transformational Learning Opportunity grants, Academic Affairs Faculty Enhancement grants, a Dean’s Leadership Council Fellowship Award, a UNF Foundation Board Grant, and research start‐up funds from the University of North Florida.
The loss of endogenous Neuregulin‐4 impairs intestinal epithelial recovery from LPS‐induced injury.
Authors: Bernard Jessica Kathleen , Schumacher Michael , Katada Kay , Fernandez Esteban , Frey Mark 
Published: April 2020

Growth factor receptors such as EGFR/erbB family members can preserve intestinal integrity by the activation of anti‐apoptotic pathways and stabilization of epithelial tight junction components. Previously we have shown that the activation of ErbB4 by its specific ligand Neuregulin‐4 (NRG4) promotes epithelial cell survival through Src and PI3 kinase/AKT pathways and reduces intestinal inflammation. However, it is unclear whether ErbB4 is an essential regulator of the intestinal barrier and if endogenous NRG4 is required for the response to intestinal inflammation. In this study we investigated the role of endogenous NRG4 as it pertains to intestinal barrier function and cell survival in vivo.METHODSNRG4 knockout mice and wild type littermates were challenged with lipopolysaccharide (LPS) injection. Intestinal permeability was determined by gavaging with 4‐kDa FITC‐dextran and collecting blood for fluorescence analysis after 4 or 24 h. The relative mRNA level of inflammatory cytokines was determined by taqman gene assays using full thickness ileal homogenates. Fixed ileal tissue for analysis of apoptosis was assessed by TUNEL and cleaved caspase‐3 staining, proliferation was evaluated by KI67 staining, and tight junction localization was determined by ZO‐1 staining.RESULTSThe loss of endogenous NRG4 in unchallenged mice did not alter baseline intestinal permeability, apoptosis, or proliferation. Interestingly, baseline inflammatory cytokine levels were reduced in the NRG4‐null mice compared to WT littermates. NRG4 KO mice showed significantly increased permeability to 4‐kDa FITC‐dextran (P<0.05) and enterocyte apoptosis (P<0.05) 24 hours after LPS‐induced injury, which was not seen 4 hours after LPS challenge. Furthermore, ZO‐1 redistribution was apparent within the base of the crypt 24 hours after LPS challenge in the NRG4 KO mice, while redistribution was only visible after 4 hours in WT mice.CONCLUSIONThe loss of endogenous NRG4 impairs the recovery of intestinal epithelial barrier after LPS‐induced injury. Further investigation of this recovery mechanism may reveal more effective strategies for targeting epithelial barrier dysfunction seen in intestinal diseases such as inflammatory bowel disease.Support or Funding Information2 R01 DK095004 06A1