bluuebunny/crossref_metadata_2025 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 Information

NIH 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 Information

This 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 Information

Juvenile 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.

METHODS

BALB/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.

RESULTS

Our 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.

CONCLUSION

This study suggests that heavy ion exposure affects skeletal muscle and can lead to discrete and combined effects in anabolic signaling.

Support or Funding Information

This 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 Information

Research 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 Information

Supported by R21 DA046044 to LCD and WK
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.

METHODS

NRG4 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.

RESULTS

The 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.

CONCLUSION

The 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 Information

2 R01 DK095004 06A1
Nrf2 transcription factor inhibition aggravates oxidative stress induced D1R dysfunction and hypertension in mice
Authors: Farooqui Zeba , Lokhandwala Mustafa F. , Banday Anees Ahmad
Published: April 2020

The renal dopamine 1 receptor (D1R) dysfunction is strongly associated with oxidative stress and high blood pressure (BP). Nuclear factor E2‐related factor 2 (Nrf2), a redox‐sensitive transcriptional factor is a key regulator in antioxidant response element (ARE)‐mediated induction of phase II antioxidant enzymes. The present study was conducted to investigate the role of Nrf2/ARE signaling pathway in renal D1R function and BP in mice. C57BL/6J mice were treated with a pro‐oxidant buthionine sulfoximine (BSO) (10 mmol/L in drinking water) and ML385, a Nrf2 inhibitor that binds to Nrf2 and inhibits its downstream target gene expression (10 mg/kg body weight/day, intraperitoneally through osmotic pumps) for 8 weeks. BSO administration to wild type mice resulted in marked increased in oxidative stress and mean arterial pressure (MAP) which was accompanied by decreased renal D1R expression and glomerular filtration rate (GFR). Also, SKF38393 (D1R agonist) infusion failed to promote sodium excretion indicating D1R dysfunction in BSO treated animals. ML385 treatment per se had no significant change in MAP, D1R, heme oxygenase‐1 (HO‐1) and NADPH quinone oxidoreductase1 (NQO‐1) expression, GFR and sodium excretion. However, co‐treatment of ML385 and BSO exhibited robust increase in MAP, aggravated oxidative damage and significantly decreased GFR. ML385 treatment to BSO administered animals further impaired the D1R function that resulted in decrease in sodium excretion. Moreover, ML385 and BSO co‐treatment showed decreased D1R and phase II antioxidant enzyme (HO‐1 and NQO‐1) expression in comparison to BSO alone animals. In conclusion, the results suggest Nrf2 inhibition exacerbated oxidative stress induced D1R dysfunction and hypertension possibly by attenuating the phase II antioxidant defense response.

Support or Funding Information

This work is funded by National Institutes of Health (National Heart, Lung, and Blood Institute) Grant HL‐139808.
Post‐partum Anxiety and Hypertension, but not Chronic Neuroinflammation Occur in Response to Hypertensive Pregnancies
Authors: Robinson ReAnna , Bowles Teylor , Solis Lucia , Dumas John P. , Spencer Shauna-Kay , Griffin Ashley , Shaffery James P. , Wallace Kedra
Published: April 2020

During pregnancy hypertensive related disorders, such as preeclampsia and HELLP syndrome, are associated with systemic inflammation and changes in the central nervous system. Previous clinical and experimental studies have shown that circulating inflammatory factors in the blood can contribute to blood brain barrier (BBB) damage in some women with these conditions, which could serve as one possible explanation for the alterations in post‐partum behavior. Using two animal models of hypertensive pregnancy (preeclampsia vs HELLP) we tested the hypothesis that T cell activation during a hypertensive pregnancy is associated with post‐partum anxiety and chronic neuroinflammation.

On gestational day (GD) 12 mini‐osmotic pumps infusing sFlt‐1 (4ug) and sEng (7ug) were placed into rats to induce HELLP or with sFlt‐1 alone to increase blood pressure. A subset of rats had 2mg/kg of Orencia infused on GD13 to decrease T cell activation. All rats delivered on GD21 and beginning postpartum week (PPW) 6 animals completed open field and zero maze testing. Blood pressure was measured via tail cuff and animals were euthanized. At euthanization brains were collected, sectioned into frontal and posterior cortices, brainstem and cerebellum. T cells and microglia were isolated from the posterior cortex and brainstem through enzymatic and microbead isolations. Cell populations were cultured overnight and assayed for inflammatory markers via ELISA. Data was analyzed via ANOVA with Tukeys multiple comparison test for post‐hoc analysis using Graphpad Prism.

All rats spent significantly less time in the open field compared to NP control rats (p<0.0001). Orencia treatment did not significantly increase the time spent in the open field compared to untreated rats (p=0.18). Both HELLP (p=0.005) and NP+sFlt‐1 (p=0.0005) rats spent significantly less time in the open sections of the zero maze compared to NP rats. However, this behavior was reversed with Orencia treatment (p=0.223). MAP was significantly increased in HELLP rats (p<0.01) and hypertensive rats (p=0.02) compared to NP rats. Orencia prevented hypertension in HELLP (p=0.01) and hypertensive rats (p=0.004) compared to the untreated rats. There were not any significant differences in IL‐17 (p=0.25 brainstem, p=0.89 posterior cortex) or TNF‐alpha (p=0.45 brainstem, p=0.11 posterior cortex) secretion between any of the brain regions or groups when analyzed via ELISA.

The results from the current study suggest that hypertension and T cell activation during pregnancy contributes to changes in post‐partum anxiety and post‐partum hypertension but not chronic neuroinflammation. Future studies will examine cognition and look at additional inflammatory pathways.

Support or Funding Information

1R01MH116027‐01A1
Changes in Bone Strength and Distal Femur Trabecular Thickness following Sleep‐Deprivation in Ovariectomized Rats Treated with Zoledronate
Authors: Nolte Erin , Frisch Frank
Published: April 2020

Osteoporosis is a disease that causes the density and quality of bone to be decreased, which can increase the risk of fracture. As bone is not a homogeneous tissue and bone mineral density (BMD) is a crude evaluation of the architectural changes seen in osteoporosis, the trabecular thickness may serve as a better single evaluation of bone health. 1 in 3 women over the age of 50 will be affected by osteoporosis. This study aimed to show how bone strength and distal femur trabecular thickness might be affected by sleep‐deprivation in estrogen‐deficient rats. Secondarily, we wanted to evaluate if Zoledronate would attenuate the deleterious consequences of sleep‐deprivation on bone. Wistar female rats were ovariectomized to contrive post‐menopausal estrogen‐deficiency. The rats were separated into 4 groups. The control group (C, n=4) was given an intraperitoneal (IP) injection of 0.45 mL of 0.9% saline, were housed in standard conditions permitting 12 hours light and 12 hours dark. The Zoledronate group (Z, n=8) were given an IP injection of 50 ug/kg of 10% Zoledronate and housed in standard conditions with a 12 hour light/dark cycle. The sleep‐deprived group (SD, n=8) were given the same IP injection as the rats in C, but were housed in chambers that did not permit sleep for 18 hours, then moved to sleep chambers for 6 hours. The sleep‐deprived Zoledronate group experienced the same sleep deprivation as the SD group, but received an IP injection of 50ug/kg body weight of 10% Zoledronate. After 5 weeks, tibiae and femora were harvested, wrapped in saline‐soaked gauze, and stored at −80F until a 3‐point bending test, high‐resolution micro‐CT and peripheral computed tomography was done. The SDZ group showed improved bone strength compared to the SD group (165.97 N and 162.21 N, respectively; N.S.). Also, the SDZ group showed improved distal femur trabecular thickness compared to the SD group (75.5 microns and 68.375 microns, respectively; p<0.05). The value of bisphosphonates in bone preservation is well documented. We attempted to illuminate the value of a bisphosphonate in sleep‐deprived environments in the hopes of evaluating the efficacy and potential therapy for executive women who consistently work in sleep‐deprived states. Our findings are equivocal but encouraging to pursue studies of a longer duration.

Support or Funding Information

Special thanks to the Novartis Pharmaceuticals Corporation for trabecular thickness analyses.
Reduced responsiveness of bone marrow hematopoietic cells to sympathetic activation is protective against high fat diet‐induced obesity and gut dysbiosis
Authors: Ahmari Niousha , Geis Natalie M. , Singh Arashdeep , de Lartigue Guillaume , Zubcevic Jasenka
Published: April 2020

INTRODUCTION

Genetic ablation of beta 1 and 2 receptors in the bone marrow (BM) results in mild immunosuppression and beneficial shifts in the gut microbiota. Considering the role of sympathetic activity, inflammation and gut dysbiosis in obesity, we hypothesized that reduction of BM responsiveness to sympathetic activation will dampen the deleterious effects of high‐fat diet (HFD) via protective host‐microbiota interaction.

METHODS

Chimera BM mice were generated by reconstituting sub lethally‐irradiated C57BL/6J mice with whole BM cells from either the beta‐adrenergic 1/2 receptor knock down (BM C57‐Adrb1.b2 KD) or C57BL/6J mice (BM C57‐C57). Circulating T cells, monocytes, and macrophages were measured at baseline. Both groups of mice were randomized to either control (ND, 10% kcal fat, n=4/5 per group) or high‐fat diet (HFD, 60% kcal fat, n=5/6 per group) ad libitum for two weeks. Body weights and food intake were monitored throughout the study. Magnetic resonance imaging was used to determine body composition and fat to muscle ratio. Visceral fat pads were weighed, and relative abundance of major gut bacterial phyla were determined using qPCR at endpoint.

RESULTS

BM C57‐Adrb1.b2 KD mice were protected from obesity‐related outcomes such as increased total weight gain, total body fat to muscle ratio, and visceral fat pad weight, all of which were all present in the BM C57‐C57 mice on HFD (P<0.05 vs ND). Interestingly, no difference in the food intake was observed between the experimental groups. BM C57‐Adrb1.b2 KD chimera also presented with lower circulating inflammatory Ly6C+ monocytes compared to the BM C57‐C57 chimera mice (p<0.05), prior to HFD feeding. Notably, BM C57‐Adrb1.b2 KD chimera were also protected from HFD‐dependent alterations in major bacterial phyla such as Firmicutes and Actinobacteria, compared to the BM C57‐C57 chimera in which the same phyla were significantly altered by HFD (P<0.05 vs ND).

CONCLUSION

These data demonstrate that the BM C57‐Adrb1.b2 KD mouse, characterized by reduced responsiveness of BM hematopoietic cells to sympathetic activation, is protected against obesity possibly via mechanisms involving reduction in pro‐inflammatory monocytes and protection against alteration in the specific gut bacterial phyla.
Right Aortic Arch with Mirror Image Branching and Situs Solitus: A case report on anatomic variation
Authors: Ensz Jordan L. , Paul Joshua , Gannon James , Bruins Anna , Carney Corey R. , Barremkala Malli
Published: April 2020

During a routine cadaver dissection performed by first year medical students at Oakland University William Beaumont School of Medicine, a 60‐year‐old female cadaver was found to have right aortic arch with mirror image branching (RAMI). The descending aorta was observed to pass to the right of the midline and subsequently to cross left, anterior to the esophagus, returning to its typical orientation at the aortic hiatus. This defect involves a mirror image branching in which a left brachiocephalic trunk, right common carotid artery, and right subclavian artery emerging directly from the aortic arch proximal to distal. The right recurrent laryngeal nerve passes under the aortic arch while the left recurrent laryngeal nerve was located more superiorly, passing underneath the left subclavian artery. The heart presented in its typical orientation with the apex located to the left of midline with no noted abnormalities of the pulmonary trunk or vascular ring surrounding the trachea. The aortic arch was studied in 24 formalin fixed cadavers (13 female, 11 male, average age 80) which had been dissected by first year medical students. Only 1 of the 24 specimens (4.17%) had a congenital aortic arch and branching defect while the remaining 23 did not display any aortic arch or branching abnormalities. After review of the published clinical literature, this variation appears consistent with RAMI and situs solitus which has been found in <0.1% of the general population. Studies have shown that 75–85% of cases with this defect are associated with cyanotic congenital heart disease, which makes early detection and follow‐up with pediatric cardiology essential. Other considerations for cardiac patients presenting with RAMI and situs solitus include accurate procedural imaging to provide safe and effective interventional care.
Iron and copper transport activities of the mammalian metal‐ion transporters DMT1 and CTR1
Authors: Azucenas Corbin R. , Shawki Ali , Logeman Brandon L. , Niespodzany Eric J. , Dunham Justin L. , Ruwe T. Alex , Thiele Dennis J. , Mackenzie Bryan
Published: April 2020

Divalent metal‐ion transporter‐1 (DMT1) and the high‐affinity copper transporter‐1 (CTR1) play essential roles in the intestinal absorption of iron and copper respectively. Both transporters are potential therapeutic targets in disorders of metal metabolism. Disagreement remains over whether the metal‐ion specificities of the two transporters overlap. For example, others have concluded that DMT1 transports Cu+ and CTR1 transports Fe2+ based on the results of shRNA knockdown of gene expression in Caco‐2 cells [Espinoza A et al (2012) Biol Trace Elem Res 146, 281]. Since these observations contradicted our published data regarding the substrate profile of DMT1 in vitro [Illing AC et al (2012) J Biol Chem 287, 30485] and in vivo [Shawki A et al (2015) Am J Physiol Gastrointest Physiol 309, G635] or of CTR1 in vitro [Lee J et al (2002) J Biol Chem 277, 4380], we have re‐examined metal‐ion transporter substrate profiles. We tested the hypothesis that DMT1 can transport copper and that CTR1 can transport iron by using radiotracer assays and the voltage clamp in RNA‐injected oocytes expressing human DMT1 or CTR1. Expression of CTR1 in oocytes stimulated up to 20‐fold the uptake of 2 μM 64Cu+ (pH 5.5) but did not stimulate the uptake of 64Cu2+ or 55Fe2+. Expression of DMT1 in oocytes stimulated 80‐fold the uptake of 2 μM 55Fe2+ (pH 6.0) but transport of 64Cu+ or 64Cu2+ did not differ from that in control oocytes. We found that Cu+ inhibited DMT1‐mediated 55Fe2+ uptake in a competitive fashion (Ki ≈ 58 μM). In DMT1‐expressing oocytes, Cu+ evoked inward currents; however, such currents were also observed in control oocytes and their I/V relationships did not resemble those for DMT1 substrates Fe2+ and Cd2+. Addition of Fe2+ or Cd2+ abolished the presteady‐state (transient) currents observed in the absence of metal ion whereas addition of Cu+ or Cu2+ did not. We found, however, that Cu2+ accelerated the decay of the transient currents. We conclude that (1) whereas Cu+ and Cu2+ are capable of interacting with DMT1, neither are transported substrates; and (2) CTR1 mediates transport of Cu+ but not of Cu2+ or Fe2+. We therefore find no evidence of overlapping metal‐ion specificities in DMT1 and CTR1.

Support or Funding Information

PHS grants R01‐DK080047, R01‐DK107309, R01‐DK074192, and R25‐HL115473; and the American Physiological Society
Identifying Direct Interactions Between the Rho GTPase TCL and Coronin CRIB Motifs
Authors: Murphy Rachel E. , Samuelson Chelsea A. , Hamann Michael J.
Published: April 2020

TCL is a Rho GTPase that contributes to tumor‐associated angiogenesis and metastatic melanoma; however, less is known about its basic cell biology and biochemistry. Currently, our lab has found that GTP‐loaded TCL tends to localize to the plasma membrane, while GDP‐loaded TCL localizes to intracellular vesicles. Our data also suggests that vesicle localization of GDP‐loaded TCL occurs through a protein/protein interaction. Coronins are a logical partner for this interaction as they bind to other Rho GTPases when they are in a GDP‐loaded form. To test for a direct interaction between Coronins and TCL, the CRIB domains from the seven human Coronin isoforms have been cloned into plasmids for use in interaction assays using both traditional GST‐fusion protein precipitation and protein complementation using an in vitro split luciferase assay system. Given the importance of TCL in angiogenesis and melanoma, delineation of its interactions with partner proteins is essential for understanding its role in pathology‐related processes.

Support or Funding Information

This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).
Myo‐Inositol Improves Ischemic Stroke Outcome After both Nicotine Containing Electronic Cigarette and Tobacco Smoke Exposure
Authors: Villalba Heidi , Vaidya Bhuvaneshwar , Cucullo Luca , Abbruscato Thomas J.
Published: April 2020

Ischemic stroke occurs in 87% of all strokes and is the leading cause of disability in the United States. Smoking has been shown to increase brain edema after ischemic stroke and worsen long‐term clinical outcomes. Electronic nicotine delivery systems or e‐cigarettes (e‐Cigs) are gaining popularity over traditional tobacco smoke cigarettes, and have been reported influence stroke outcome. Previous work from our lab reported nicotine and e‐Cig exposure decreased glucose utilization in the brain in ischemic stroke models, further exacerbating the effects of ischemic injury. Myo‐inositol(MI), a major organic osmolyte that promotes cell membrane stability and various cellular functions, has been reported to reduce infarcted area and edema ratio in a diabetic ischemic mouse model. We also sought to understand the role of MI protection with regard to additional stroke comorbidities, such as e‐Cig or tobacco smoke exposure. In this study, we investigated the neuroprotective role of MI transporters during stroke and post‐stroke outcomes, especially related to the role of glial cells and post‐stroke reorganization. We hypothesize MI plays a role in increased recovery of motor function, after stroke, in animal groups exposed to tobacco smoke or e‐Cig vape. Primary astrocytes and primary neurons were used for in vitro studies and a middle cerebral artery occlusion (MCAO) mouse model for in vivo stroke studies. Immunocytochemistry (ICC) studies showed astrocytes express sodium dependent myo‐inositol transporter 1 (SMIT1) and sodium‐dependent glucose transporter 6 (SGLT6). Our data illustrated expression of both transporters increased in a time‐dependent manner during oxygen‐glucose deprivation (OGD), in tobacco smoke extract (TSE), and e‐Cig extract (ECE) treated cells. Further, this was confirmed by an increase of MI uptake in TSE treated astrocytes. Altered cell mitochondrial respiration levels were observed in astrocytes and neurons incubated for 24hr with TSE and ECE followed by OGD exposure using Seahorse XF Cell Mito Stress. Animal studies demonstrated post‐stroke administration of MI, reduced infarction size and edema ratios in tobacco smoke compared to non‐tobacco smoke animals. An improvement of motor function was also seen in selected behavioral tests. E‐cig exposed animals had a slight increase in infarction size compared to non‐e‐Cig animals. This preliminary data suggest myo‐inositol may have a neuroprotective role in ischemic stroke injury and could significantly improve stroke outcome in both tobacco smokers and e‐Cig users. Further investigation will include assessment of sex differences with regard to the neuroprotective mechanisms of MI in e‐Cig and tobacco smoke exposed animals.

Support or Funding Information

1R01DA0497375 and R01DA029121 to TJA and LC
The Potential Impact of Discrete Action Potentials on Resting Sympathetic Vascular Transduction in Humans
Authors: Cloud Jane N. , Young Benjamin E. , Greaney Jody L. , Shoemaker J. Kevin , Fadel Paul J.
Published: April 2020

The sympathetic nervous system dynamically regulates blood pressure (BP), primarily through the modification of peripheral vascular tone. Our lab has developed a methodology for quantifying resting sympathetic vascular transduction wherein bursts of muscle sympathetic nerve activity (MSNA) act as a trigger and ensuing changes in BP and vascular conductance are signal‐averaged over the subsequent 15 cardiac cycles to produce characteristic (mean) responses. These previous investigations have focused on the effect of net sympathetic outflow (e.g, burst sequences and burst amplitudes) on the pressor and vascular conductance responses to spontaneous bursts of MSNA. The recent development of methods to identify sympathetic action potentials (APs) to obtain further information regarding sympathetic control of the circulation has led us to begin to examine the potential role that discrete sympathetic APs may play in determining the BP and vascular responses following spontaneous bursts of MSNA. Herein, we compared BP and leg vascular conductance responses between MSNA bursts containing small (i.e., common) or large (i.e., uncommon) APs within quartiles of MSNA normalized to burst amplitude. We hypothesized the presence of large APs in a burst of MSNA would elicit a greater pressor response and a larger decrease in leg vascular conductance. Resting MSNA (microneurography), beat‐to‐beat BP (finger photoplethysmography), and femoral artery blood velocity (Doppler ultrasound) were collected for 10–20 minutes in 5 young healthy adults. Leg vascular conductance index was calculated (LVCi=femoral artery blood velocity/mean arterial pressure). MSNA bursts were sorted into normalized amplitude quartiles (Q1=smallest 25% of bursts; Q4=largest 25% of bursts), and APs were extracted using a matched wavelet methodology. Within Q4, there was no difference in the peak BP or LVCi responses to bursts containing small APs when compared to bursts containing large APs (Δ6±3 small vs. Δ6±2 large mmHg, p=0.74; Δ0.95±2.0 small vs. Δ0.87±1.7 large ml·min−1·mmHg−1, p=0.59). To account for the potential contribution of MSNA bursts occurring in consecutive sequence, we next isolated singlet bursts (separated by ≥1 cardiac cycle lacking MSNA bursts). Within Q4, there was again no difference in the peak BP or LVCi response to singlet bursts containing small APs compared to singlet bursts containing large APs (Q4: Δ7±3 small vs. Δ6±2 large mmHg, p=0.42; Δ0.05±0.07 small vs. Δ0.17±0.11 large ml·min−1·mmHg−1, p=0.08). These preliminary data suggest that the presence of large APs does not appear to translate to greater pressor or decreased leg vascular conductance responses following a spontaneous burst of resting MSNA.

Support or Funding Information

Funded by the UTA College of Nursing and Health Innovation and Natural Sciences and Engineering Research Council of Canada
The soluble form of the prorenin receptor disturbed glucose/insulin homeostasis and leptin secretion in obese male mice
Authors: Poupeau Audrey , Gatineau Eva , Yiannikouris Frederique
Published: April 2020

Obesity is one of the major causes of the rise in prevalence of the metabolic syndrome and hypertension. We recently found that the circulating form of the prorenin receptor (sPRR) increased during the development of obesity. Interestingly, in high‐fat fed C57BL/6 male mice, the infusion of sPRR increased systolic blood pressure through an impairment of the baroreflex sensitivity and the increase in the sympathetic tone, potentially mediated by a leptin‐dependent mechanism. In the present study, we aim to address the contribution of sPRR to the development of the metabolic syndrome in obesity.

We first investigated whether sPRR plays a role in glucose and insulin (G‐I) homeostasis. C57BL\6 male mice were fed a high fat (HF) diet for 16 weeks. On week 17 of HF feeding, mice were implanted with an osmotic pump infused for 4 weeks with saline (Veh, N=7) or sPRR (mouse recombinant sPRR‐HisTag, 30 μg/kg/day) (sPRR, N=7). After 12 days of infusion, fasting glucose were examined and intraperitoneal glucose and insulin tolerance tests were performed. Fasting glucose levels were significantly higher in HF‐fed mice infused with sPRR compared with HF‐fed mice infused with vehicle (Glucose: Veh: 180±8 mg/dl; sPRR: 205±5 mg/dl. P<0.05). In addition, HF‐fed mice infused with sPRR exhibited impaired glucose tolerance compared with HF‐fed mice infused with vehicle (area under the curve: Veh: 27143 ± 3210; sPRR: 34702±4191. P<0.05). Despite a higher glucose levels after insulin injection, HF‐fed mice infused with sPRR had a similar reduction in blood glucose in response to insulin compared with HF‐fed mice infused with vehicle. Interestingly, the phosphorylation levels of hepatic Akt decreased significantly in HF‐fed mice infused with sPRR compared with vehicle (p‐Akt/Akt ratio: Veh: 1.0±0.1, sPRR 0.4±0.1 AU, P<0.05) suggesting that sPRR might impair hepatic insulin sensitivity. Downstream glucose and lipid metabolic pathways are currently under investigation in liver and peripheral tissues.

Since circulating leptin were elevated in HF‐fed mice infused with sPRR, we next aimed to determine the relative contribution of sPRR to leptin secretion. In vitro studies demonstrated that leptin secretion increased significantly in differentiated 3T3‐L1 cells treated with sPRR compared with cells treated with vehicle indicating that sPRR directly stimulated adipocyte release of leptin and could participate to leptin resistance in obesity.

Together our data indicated that sPRR likely worsen the metabolic syndrome by affecting G‐I homeostasis and by stimulating leptin secretion.

Support or Funding Information

This work was supported by National Institutes of Health Grants [R01‐HL‐142969]; the American Heart Association [13SDG17230008]; the National Institute of General Medical Sciences [P30 GM127211]; and the University of Kentucky, Center for Clinical and Translational Sciences [UL1TR001998].
The Effects of N‐terminal Acetylation on the Protein Tau
Authors: Fleurima Abigail , Holmes William
Published: April 2020

The microtubule‐associated protein tau (MAPT) plays a critical role in many neurodegenerative diseases such as Alzheimer’s Disease. Tau functions to stabilize microtubule structures that are essential for transport within the neuron, and disruption of transport leads to a loss of neuronal function. Tau binding is regulated by phosphorylation, a post‐translational modification (PTM) where a phosphate group is added by a specific kinase. Phosphorylated Tau blocks the microtubule binding sites, then phosphatases remove these phosphate groups which expose the microtubule‐binding regions. Tau is also found in a hyper‐phosphorylated state, which causes Tau to self‐assemble into oligomers and higher order aggregates. It's clear that post‐translational modification of Tau plays a key role in the dysregulation of neuronal function due to abnormal conformational changes, however not all PTMs are as thoroughly studied as phosphorylation.

However, N‐terminal acetylation is the most common and overlooked PTM of all protein, this is adjacent to the proven idea of Tau being a predicted target of N‐terminal acetylation. N‐terminal acetylation is a co‐translational process that is catalyzed by N‐terminal Acetyltransferases (NATs), which adds an acetyl group to the N‐terminus of a Tau polypeptide, thus neutralizing the positive charge on the N‐terminus.

The objective of this research is to understand how the N‐terminal modification of a Tau polypeptide effects the behavior of Tau and its aggregation. Commonly, studies endogenously express Tau in a prokaryotic system that lack PTMs. Here we utilize a co‐expression system in E. coli that allows us to purify Tau with a modified N‐terminus. Purifying modified Tau with Fast Protein Liquid Chromatography (FPLC) will allow us to probe the structural and functional effects of N‐terminal acetylation by assessing changes in Tau’s aggregation and microtubule binding affinity. Furthermore, our studies of the N‐terminus of Tau will provide insight into the role of the N‐terminal projection domain and potentially provide new targets for therapeutic intervention in the treatment of Tau related diseases.

Support or Funding Information

Collaborative Award through RI‐INBRE from the National Institute of General Medical Sciences of the National Institute of Health under grant #P20GM103430.