bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinically‐Tested SOD Mimic, MnTnBuOE‐2‐PyP5+, Acutely Decreases Blood Pressure via Sympathoinhibition and Vasodilation
Authors: Schlichte Sarah L. , Park Song-Young , Kosmacek Elizabeth A. , Katsurada Kenichi , Patel Kaushik P. , Oberley-Deegan Rebecca E. , Zimmerman Matthew C.
Published: April 2020

It is well‐established that reactive oxygen species (ROS), particularly superoxide (O2•−), produced in the vasculature and brain contribute to the pathogenesis of hypertension. We, and others, have previously reported that in vivo scavenging of O2·− via overexpression of superoxide dismutase (SOD) protein or SOD mimics decreases blood pressure in hypertensive animals. While few SOD mimics have transitioned to clinical trials, MnTnBuOE‐2‐PyP5+ (BuOE), a manganese porphyrin SOD mimic, is currently in clinical trials for normal tissue protection in cancer patients exposed to radiation therapy, and thus, provides hope for the use of SOD mimics in the clinical setting. Considering the extensive evidence indicating that scavenging of O2•− decreases hypertensive blood pressures, we tested the hypothesis that the SOD mimic, BuOE, decreases blood pressure in normotensive and angiotensin II (AngII)‐induced hypertensive mice. First, using electron paramagnetic resonance (EPR) spectroscopy and hypoxanthine (HX) + xanthine oxidase (XO) to generate O2•− in a cell‐free system, we confirmed the ability of BuOE to scavenge O2•−. Superoxide produced by HX + XO yielded an EPR spectrum amplitude of 5.6×106 ± 1.3×105 EPR arbitrary units (au), which was significantly (p < 0.05) attenuated by 50 nM BuOE (3.3×106 ± 4.2×104 au). In normotensive and AngII‐induced hypertensive mice, BuOE (1 mg/kg, IP), significantly (p<0.05) decreased mean arterial pressure (MAP), as measured by radiotelemetry, within 25 minutes post‐injection (normotensive ⊗ MAP: vehicle −3.3 ± 2.8; BuOE −27 ± 5.4 mmHg; hypertensive ⊗ MAP: vehicle 3.1 ± 7.2; BuOE −60 ± 14 mmHg). To explore the mechanism(s) by which BuOE decreases MAP, renal sympathetic nerve activity (RSNA) was recorded in normotensive rats injected with BuOE (0.1 mg/kg, IV). Immediately following BuOE injection, RSNA significantly (p < 0.01) decreased by 35 ± 7.0% of baseline, followed by a significant (p < 0.01) decrease in MAP (46 ± 13 mmHg). Additionally, to explore the impact of BuOE on vascular reactivity, video myography was performed on isolated skeletal muscle arterioles from normotensive rats. BuOE (2 μM) induced significant (p < 0.05) vasodilation (31 ± 10%) that was attenuated by pretreating arterioles with the endothelial nitric oxide synthase (eNOS) inhibitor, L‐NAME (1 mM); thus, suggesting that enhanced nitric oxide bioavailability mediates BuOE‐induced vasodilation. Collectively, these data indicate that BuOE, a SOD mimic currently in clinical trials in cancer patients, acutely decreases blood pressure in both normotensive and hypertensive conditions by decreasing sympathetic nerve activity and inducing vasodilation.

Support or Funding Information

University of Nebraska Medical Center Graduate Student Assistantship/Fellowship
Structural Characterization of Histone Octamers at Varied Salt Concentrations Using Hydrogen‐Deuterium Exchange Mass Spectrometry
Authors: Timmerman Adam
Published: April 2020

Histones, the positively‐charged core proteins that form nucleosomes and aid compaction and organization of DNA in eukaryotic organisms, come together to form an octamer in the presence of negatively‐charged DNA. The charge stabilization provided by DNA can be artificially replicated by introducing the proteins to a high salt environment. Hydrogen‐deuterium exchange mass spectrometry was used to characterize histone‐histone interaction stability as a function of salt, with octamers readily breaking down into constituent histones at lower salt concentrations. The relative uptake of deuterium in both solitary histone and fully‐formed octamer samples after various time points revealed the exposure of different parts of the proteins to the surrounding buffer, demonstrating when the histone‐histone interactions were destabilized. Stable octamer interactions were only observed over a long period of time at 2M salt, and their stability was optimized in a separate experiment, conducted at 4°C, at 2M salt as well.
An Allosteric Site Residue Critical for Transmission of the Allosteric Signal of the Vibrio cholerae SpeG Enzyme
Authors: Marquez-Ramirez Martha , Tran Ha , Kuhn Misty L.
Published: April 2020

SpeG is a polyamine N‐acetyltransferase enzyme that belongs to the superfamily of Gcn5‐related N‐acetyltransferases (GNATs). We previously showed this enzyme from Vibrio cholerae (VcSpeG) acetylates spermine and spermidine. SpeG is thought to contribute to biofilm formation in several pathogenic bacteria and several studies have shown that it also plays a role in bacterial polyamine homeostasis under stressful conditions. This GNAT is unique because it forms a dodecameric structure and has an allosteric site in each monomer. Additionally, it binds polyamines at both allosteric and active sites. Previous X‐ray crystallographic studies have shown that the E41 residue in the allosteric site of VcSpeG forms a hydrogen bond with N10 of spermine and N6 of spermidine. We hypothesized that mutating this residue would affect the allosteric network of the enzyme. Therefore, we created a series of E41 point mutations and investigated the enzymatic activity and kinetic parameters toward spermine and spermidine. Changing the identity of the residue at this location either increased the catalytic efficiency and altered the sigmoidicity of the substrate saturation curve or decreased the catalytic efficiency and affected cooperativity. This implies that the identity of the residue at this position affects the allosteric signal that is transmitted. Further investigation into other residues in this site is warranted.

Support or Funding Information

Research reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM133506 (to MLK).
Therapeutic Effect of Oridonin Against Inflammatory Bowel Disease Involves Inhibition of Intestinal Fibrosis via NF‐κB Pathway
Authors: Wang Xiaofu , Cummins Claire , Gu Yanping , Song Juquan , Zhou Jia , Radhakrishnan Ravi
Published: April 2020

Introduction

Intestinal fibrosis and stricture formation are frequent complications of inflammatory bowel disease (IBD). In response to inflammatory stimuli, intestinal epithelial cells may undergo epithelial‐mesenchymal transition (EMT). EMT is a process by which epithelial cells acquire a mesenchymal‐like phenotype, resulting in intestinal fibrosis. Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, was reported to have therapeutic effects for IBD. However, the cellular mechanism remains unclear. We hypothesize that oridonin attenuates inflammation‐related EMT and fibrogenesis in intestinal epithelial cells.

Methods

IEC‐6 and IEC‐18 cells exposed to TNFα were pre‐treated with or without oridonin. Cellular proteins were analyzed with Western blot or immunofluorescence assay. Cytokine secretion was determined with ELISA.

Results

Oridonin treatment suppressed TNFα‐induced NF‐κB activation evidenced by blocking TNFα‐induced NF‐κB p65 nuclear translocation and DNA binding activity. TNFα‐induced phosphorylation of IKKα/β and IκBα as well as total IκBα degradation were prevented by oridonin. TNFα‐stimulated secretion of pro‐inflammatory cytokine IL‐6 was inhibited by oridonin. Oridonin moderately up‐regulates endogenous E‐cadherin and down‐regulates Snail. The expression of extracellular matrix proteins fibronectin and laminin were increased by TNFα and suppressed by pre‐treatment with oridonin. For the first time, we demonstrated that TNFα dose‐dependently up‐regulated α‐smooth muscle actin (α‐SMA), a myofibroblast marker, in IEC‐6 cells. These results indicate that IEC‐6 cells underwent transition into mesenchymal cells after TNFα stimulation. Notably, oridonin inhibited TNFα‐stimulated α‐SMA, but not TGFβ1‐stimulated EMT‐related proteins.

Conclusion

The protective effects of oridonin in intestinal epithelial cells may occur through suppression of the TNFα‐induced NF‐κB pathway and EMT‐related fibrogenesis.
SPARC ‐ Mapping Gut‐Spinal Cord Connections in Visceral Pain
Authors: Morrie Ryan David , Bayrer James , Brierley Stuart , Ingraham Holly , Julius David
Published: April 2020

Our current understanding of mechanisms underlying visceral pain, including that associated with irritable bowel syndrome, remains rudimentary. Importantly, opiates are ineffective at treating visceral pain syndromes, and only exacerbate discomfort by producing constipation, reflecting a clear need for alternative treatment options. Our goal is to bring greater mechanistic insight to this underserved area of pain research, and to approach the problem in a multifaceted strategy designed to maximize the relevance of our basic research discoveries to future pain treatments. Specifically, we are asking how enterochromaffin (EC) cells transmit noxious signals from the gut lumen to the spinal cord. EC cells are key sensory cells in the intestinal epithelium that release serotonin onto primary sensory nerve fibers, thereby evoking a sensation of discomfort and pain in response to luminal irritants, such as bacterial metabolites, inflammatory agents, or ingested chemicals. We are using transgenic moue models to selectively activate and silence EC cells to examine their functional connections with sensory nerve fibers and spinal circuits. We are coupling these tools with transcriptome profiling, viral tracing, and electrophysiological methods to gain insights into the molecular and functional identity of these fibers.

Support or Funding Information

NIH R35 NS105038, NIH U01 NS113869, Rainin Foundation, Giannini Foundation
Neck Muscle Morphology and Function in Primates
Authors: McGechie Faye , Grider-Potter Neysa , Nalley Thierra , Fricano Elle , Ward Carol
Published: April 2020

The cervical spine lies at intersection between the cranium and postcranium and plays a crucial role in orienting the head relative to the substrate during locomotion. Previous work demonstrates that bony features of the cranial base and cervical spine are related to positional behavior in primates. How soft tissues of the neck vary across primate taxa is relatively unknown yet is critical for understanding function, head carriage, and locomotion in this clade. The objective of this study was to describe the relationship between the extrinsic and intrinsic muscles of the neck and postural and locomotor behaviors in primates. We collected and dissected 26 primate cadavers from 17 different genera representing each major primate clade. We hypothesized that trunk and neck posture, as well as forelimb dominated below‐branch suspensory behaviors and neck rotational capabilities all influence the attachment sites and size of the dorsal neck musculature.

Using gross anatomical dissection, we qualitatively described and compared the attachment sites of the dorsal neck, back, and shoulder muscles of taxa that display contrasting behaviors and quantified muscle mass. Using the dissection data combined with contrast enhanced microCT data, we constructed 3D models of the head and neck to visualize the in situ neck muscle attachment sites for Propithecus, Aotus, and Carlito.

Gross anatomical dissections demonstrate that among nonhuman apes, hylobatids lack cranial attachments of the trapezius and rhomboideus. Additionally, strepsirrhines display anatomy that is more nonprimate mammal‐like in the distribution of the trapezius, rhomboideus, and semispinalis muscles. Results of phylogenetic regressions indicate that neck muscle masses are best predicted by body size, but also demonstrated significant results for both postural and locomotor behaviors as predictors of overall muscle mass (p < 0.05). This study represents the first comprehensive description of the functional anatomy of the neck muscles across primates. Future work will focus on quantifying biomechanically‐relevant variables such as physiological cross‐sectional area using novel fascicle‐tracking methods and estimating muscle moments to compare across taxa.

Support or Funding Information

This work was funded by the Leakey Foundation, National Science Foundation, American Association of Anatomists, Western University of Health Sciences, and University of Missouri.
Consequences of maternal bariatric surgery: potential for reduced amino acid availability to the fetus
Authors: Welch Bradley A. , Himel Alexandra , Duncan Brittany , Spann Redin A. , Grayson Bernadette E.
Published: April 2020

Though bariatric surgery is the most effect method of durable weight loss, it is not understood why or how it works so effectively to reduce the comorbidities of Metabolic Syndrome. Both in humans and rodents, maternal bariatric surgery prior to pregnancy results in challenges during pregnancy including increased small‐for‐gestational age offspring and greater risk for fetal demise. We have shown in the rodent model of vertical sleeve gastrectomy (VSG) that offspring develop greater risk for metabolic disease later in life compared to obese controls. A common gestational cause of poor growth in utero is maternal protein deficiency. In the current work, we hypothesize that the transporters within the placenta may be differentially transporting amino acids to the fetus despite previously demonstrating that protein levels in the VSG dam were not harmed by surgery and were sufficient to sustain a health pregnancy. Using gestational day 19 placenta from pregnant Lean, Obese and post‐VSG dams, we performed analysis of the amino acid content of the placenta and prepared placental mRNA for quantitative PCR analysis of genes associated with amino acid transport. Aliphatic amino acids: isoleucine, leucine, proline and valine were significantly reduced in comparison to Lean dam levels (p< 0.05). Phenylalanine, aspartic acid and glutamic acid, arginine, histidine and methionine were also significantly reduced (p< 0.05). Furthermore, amino acid transporter SLC38A1 (SNAT1) and SLC38A4 (SNAT4) showed trends towards reductions in both VSG and Obese dams. On the contrary, SLC38A2 (SNAT2) and SLC7a7 (LAT1) was significantly elevated (p< 0.05) in VSG in comparison to Lean and Obese. This affect appeared specific to amino acids since carbohydrate transporters remained unchanged. Collectively, these data suggest that despite healthy ingestion of proteins by the VSG dam, alterations in amino acid availability to the fetus may be obstructed by the placenta after bariatric surgery. This reduced level of amino acid availability may contribute to poor outcomes in bariatric pregnancy. More work is needed to determine the relevance of these findings to humans.

Support or Funding Information

Research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM104357 and P20GM121334.
Effect of Superior Cervical Gangionectomy on the Chemoreflex Response to Hypoxia in the Rat Carotid Body
Authors: Getsy Paulina M. , Coffee Gregory A. , Liu Yehe , Owens Joy , Jenkins Michael W. , Lewis Stephen J.
Published: April 2020

Carotid body (CB) chemosensitive glomus (type I) cells respond to decreases in blood pO2 (hypoxia) by releasing neurotransmitters, which in turn activate adjacent chemosensory afferent fibers within the carotid sinus nerve (CSN). These CSN chemoafferents then signal to the commissural nucleus tractus solitarius (cNTS) within the dorsal brainstem, which leads to a compensatory increase in ventilation in order to restore blood‐gas homeostasis. The CB receives sympathetic innervation via the ganglioglomerular nerve (GGN), whose cell bodies reside in the superior cervical ganglion (SCG). We have found that electrical stimulation of the GGN causes an increase in CB chemosensory afferent discharge. However, it is unknown the effect removing the carotid body’s sympathetic innervation, via superior cervical ganglionectomy, will have on the carotid body’s chemoreflex response to hypoxia. We hypothesize that loss of sympathetic input will decrease the hypoxic ventilatory response (HVR) of the rat, and alter the O2‐sensing mechanism of the CB chemosensitive glomus cells. Sprague Dawley male rats postnatal day 21 were subjected to unilateral or bilateral superior cervical ganglionectomy (SCGX). Four days post surgery (post‐natal day 25), rats either underwent whole body plethysmography recordings or were sacrificed and their carotid bodies removed to either isolate glomus cells and perform whole cell perforated patch clamp, or perform immunohistochemistry on the CB. Our preliminary results show that unilateral (left or right) SCGX appears to decrease the HVR during a 1‐minute exposure to hypoxia (10% O2, 90% N2) compared to bilateral SCGX. Our results also show that following SCGX, isolated glomus cells responded to acute hypoxia (HX) (5–8% O2, 5% CO2, balanced N2) with significant reductions (P<0.05 to P<0.01) in outward K+ current amplitudes over the test potential range +10 to +60 mV. The mean maximal effect, a reduction in K+ amplitude of 64.9 ± 18.5% (mean ± SD, n = 3 cells), occurred at a Vtest of +30mV. In contrast, control carotid body glomus cells responded to acute HX with a significant reduction (P<0.05) in outward K+ current amplitude at a Vtest of +60mV (n = 4 cells). In conclusion, our data suggests that sympathetic innervation to the CB is critical for the ventilatory response to hypoxia, and the O2‐sensing mechanism at the level of the CB glomus cells. Overall this study provides novel evidence for the importance of sympathetic innervation on peripheral chemosensors during exposure to hypoxia.

Support or Funding Information

This work was supported in part by a SPARC NIH award OT2OD023860
Human Red Blood Cell Uptake and Sequestration of Arsenite and Selenite: Evidence for the Formation of a Protective Glutathione Conjugate
Authors: Leslie Elaine M. , Kaur Gurnit , Javed Warda , Ponomarenko Olena , Swanlund Diane P. , Hu Michael , Summers Kelly L. , Casini Angela , Wenzel Margot , Casey Joseph R. , Cordat Emmanuelle , Pickering Ingrid J. , George Graham N.
Published: April 2020

Over 200 million people worldwide are exposed to the proven human carcinogen, arsenic, in contaminated drinking water. In laboratory animals, arsenic and the essential trace element, selenium, can undergo mutual detoxification through the formation of the seleno‐bis(S‐glutathionyl) arsinium ion [(GS)2AsSe]−, which undergoes biliary and fecal elimination. [(GS)2AsSe]−, formed in animal red blood cells (RBCs), sequesters arsenic and selenium, and likely slows the distribution of both metalloids to the liver, and other organs susceptible to toxic effects. In human RBCs, the influence of arsenic on selenium accumulation, and vice versa, is largely unknown. The aims of this study were to characterize arsenite (AsIII) and selenite (SeIV) uptake by human RBCs, to determine if SeIV and AsIII increase the respective accumulation of the other in human RBCs, and ultimately to determine if this occurs through the formation and sequestration of [(GS)2AsSe]−. 75SeIV accumulation was inhibited by 4,4′‐diisothiocyanatodihydrostilbene‐2,2′‐disulfonic acid (H2DIDS) (IC50 1 ± 0.2 μM), suggesting uptake is mediated by the erythrocyte anion‐exchanger 1 (AE1 or Band 3, gene SLC4A1). HEK293 cells overexpressing AE1 showed concentration‐dependent 75SeIV uptake. 73AsIII uptake by human RBCs was temperature‐dependent and partly reduced by aquaglyceroporin 3 inhibitors. AsIII increased 75SeIV accumulation (in the presence of albumin) and SeIV increased 73AsIII accumulation in human RBCs. Near‐edge X‐ray absorption spectroscopy revealed the formation of [(GS)2AsSe]− in human RBCs exposed to both AsIII and SeIV. The sequestration of [(GS)2AsSe]− in human RBCs potentially slows arsenic distribution to susceptible tissues and could reduce arsenic‐induced disease.

Support or Funding Information

Canadian Institutes of Health Research, Canada Research Chairs program, Natural Science and Engineering Council of Canada
Barreling Forward: Advancements in De Novo Protein Design
Authors: Kohli Khushi , Liu Samuel , Garapaty Arjun , Dai Eddie
Published: April 2020

Natural evolution and protein redesign have only explored a tiny fraction of theoretically possible protein structures. However, de novo protein design will prove powerful in engineering novel proteins with specific functions by comprehensively sampling the protein space using computational methods guided by protein folding principles. De novo protein design involves working backwards from a desired protein with special structure and function to derive an amino acid sequence capable of folding into such a conformation—a challenging approach due to the vast number of protein conformations to evaluate. Here, we present a review of the latest advancements in de novo protein design methods and applications in conjunction with a physical model of a de novo fluorescence‐activating β‐barrel protein. Approaches to de novo protein design include: (1) simulating the folding of a fixed amino acid sequence to determine each residue’s most stable conformation, (2) determining the amino acid sequence that folds into a desired protein conformation, and (3) performing comprehensive rapid evaluations of each possible conformation. This novel protein is then synthesized and validated through in vitro or in vivo assays. In one recent breakthrough by Baker et. al., a de novo approach was used to design a β‐barrel protein comprised of a cylindrical pleated β‐sheet which binds the fluorogenic compound DFHBI. This study is the first instance of a de novo protein designed to bind to a small molecule of interest, with applications in visualizing cell movement, gene expression, DNA replication, protein translation, and tumor progression. In designing this fluorescence activating β‐barrel protein, structural irregularities were introduced to stabilize the structure and enlarge the β‐barrel cavity. Subsequently, the optimal sequences for β‐barrels with reasonable binding site and affinity for DFHBI were designed by computationally adjusting individual residue conformations. Finally, this optimized protein was synthesized, which had greater affinity with DFHBI and increased fluorescence in in vitro and in vivo experiments.

Support or Funding Information

This is a SMART Team project supported through the contributions of Dr. Jinan Wang of the University of Kansas Center for Computational Biology, the Milwaukee School of Engineering, and the Olathe Medical Professions 21st Century Academy.
Kidney alkalization increases macula densa NOS1β expression and improves transplanted renal graft function
Authors: Wang Lei
Published: April 2020

Ischemia‐reperfusion injury (IRI) is a major hurdle for the survival of transplanted grafts. Currently, there are no effective strategies for prevention and treatment of IRI during kidney transplantation (KTX). Neuronal NO synthase beta (NOS1β) is the primary splice variant of NOS1 in macula densa (MD) and contributes to most of the MD‐NO generation, which blunts tubuloglomerular feedback (TGF) response and increase GFR, which is one of the important factors in evaluation of renal function. While TGF response modulated by NOS1β has been demonstrated to play an essential role in the long‐term control of hemodynamics, the role of NOS1 in the MD in the prevention of IRI and protection of transplanted graft function remains unknown.

KTX was performed in C57BL/6 mice. The expression of NOS1 and graft function were evaluated. To test the response of NOS1 to the tubular pH, the mice were given bicarbonate or NH4Cl for two weeks. Blood, urine pH and expression of NOS1 were measured. KTX were performed after the treatment. Graft injury and function were evaluated again. To further investigate the role of the NOS1 in the protection of the graft function, we repeated the above experiments in kidney specific NOS1 knockout (KO) mice and compared the results with C57BL/6 mice.

The control recipients showed moderate graft injury with plasma creatinine (Pcr) of 0.68 ± 0.13 mg/dl and reduced graft function indicated by 23% decrease in GFR at 5 days after KTX. NOS1β expression decreased by 60%. TGF response was measured by micropuncture which was 4.1 ± 0.3 mmHg for sham‐operated mice and 7.2 ± 0.9 mmHg for KTX mice. These data indicated that decreased NOS1β expression may contribute to the decrease of GFR and deterioration in graft function by enhanced TGF response. KO recipients showed more severe graft injury with the Pcr of 1.07±0.14mg/dl which demonstrated that deletion of NOS1 from macula densa significantly aggravated renal graft injury.

The pH‐NOS activity response curve showed that NOS1 was sensitive to tubular pH with the optimal pH of 8.0. Bicarbonate treatment increased NOS1 expression in macula densa by 65% while NH4Cl intake decreased the NOS1 expression by 40%.

KTX were performed in C57BL/6 and KO mice after two weeks treatment of bicarbonate or NH4Cl. Pcr levels were about 30% and 100% lower in bicarbonate‐treated group than H2O and NH4Cl groups, respectively, indicating that bicarbonate treatment significantly alleviated graft injury. However, there were no significant differences in the graft injury for the KO mice among different groups.

In summary, NOS1β expression in the macula densa decreases after KTX, which enhances TGF responsiveness and contributes to the decrease in GFR and decline of graft function. Rescue of NOS1β in the macula densa by renal alkalization decreases kidney injury and improves transplanted graft functions.
The role of the rostral ventrolateral medulla in systolic blood pressure frequency content within the Mayer wave band
Authors: Badenhorst Cecilia , Squair Jordan W. , Gautier Matthieu , Soriano Jan Elaine , Courtine Gregoire , Phillips Aaron
Published: April 2020

A primary supraspinal structure involved in sympathetically‐mediated blood pressure control is the rostral ventrolateral medulla (RVLM). The RVLM is where the cell‐bodies of descending sympatho‐excitatory axons responsible for blood pressure control are located. The frequency through which the sympathetic nervous system modulates blood pressure has been termed the Mayer wave. The precise origin of Mayer waves has never been clearly identified.

Using a rat model, our objective was to assess the causal role of the RVLM in Mayer waves, which in rats occur in the low‐frequency range of 0.4–1.0 Hz. We tested this by assessing wavelet spectrograms in response to 1) optogenetic stimulation of the RVLM, 2) orthostatic activation of the RVLM and 3) surgical disruption of descending sympatho‐excitatory pathways. We optogenetically stimulated tyrosine hydroxylase (TH)ON neurons of the RVLM in a TH:Cre rat and found increased power in the Mayer wave band of the wavelet spectrogram. When we applied an orthostatic challenge to the rodents, in order to increase baroreflex‐mediated RVLM activity, the increased power in the wavelet spectrogram was again observed. In contrast, we did not find this effect in rats after a severe T3 spinal cord contusion, which disrupts descending sympatho‐excitatory pathways. In conclusion, THON neurons in the RVLM appear to be responsible for systolic blood pressure frequency content in the Mayer wave band.

Support or Funding Information

Canadian Institutes of Health Research, Libin Cardiovascular Institute, Hotchkiss Brain Institute, Wings for Life, Campus Alberta Neuroscience.
Glutathione Peroxidase Influence on Cardiac Remodeling in Progeny of Dams Exposed to Engineered Nanomaterials during Gestation
Authors: Kunovac Amina , Hathaway Quincy A. , Taylor Andrew D. , Durr Andrya J. , Goldsmith William T. , Pinti Mark V. , Fink Garrett K. , Nurkiewicz Timothy R. , Hollander John M.
Published: April 2020

Vulnerable populations, like the developing fetus, may be more susceptible to sustained detriments elicited by inhaled engineered nanomaterials (ENMs) entering the bloodstream and prompting systemic effects. Previous studies have demonstrated diminished mitochondrial bioenergetics in progeny following maternal exposure to a highly utilized ENM, nano‐titanium dioxide (nano‐TiO2). Cardiac dysfunction and reduced ROS scavenging ability were observed with decreased mitochondria phospholipid hydroperoxide glutathione peroxidase (mPHGPx) expression. This study aimed to elucidate whether overexpression of mPHGPx could provide sufficient antioxidant protection to attenuate cardiac dysfunction in pregnant dams and progeny following gestational ENM inhalation exposure. Wild‐type (WT) and transgenic (mPHGPx) female mice were bred with transgenic and WT males, respectively, to delineate maternal vs progeny protection. Pregnant dams were exposed to nano‐TiO2 (6 hrs/day for 6 days) with a primary particle size of 21nm and mass concentration of 12mg/m3 or sham air. Conventional (M‐Mode) and speckle tracking stress‐strain echocardiographic parameters were assessed in pregnant dams, as well as in fetal (gestational day 14) and adult (11 wks) offspring. Following inhalation exposure to nano‐TiO2 during gestation, WT dams exhibited signs of concentric remodeling marked by significantly decreased (left ventricular) LV diastolic diameter (12%) and volume (26%), and significant increases in both LV posterior wall thickness (23%) and relative wall thickness (39%), which were preserved in mPHGPx pregnant dams. Fetal offspring of mPHGPx dams had significantly enhanced cardiac output (87%) and stroke volume (43%) compared to fetal offspring of WT dams. Stress‐strain analysis revealed that fetal WT offspring of nano‐TiO2–exposed mPHGPx dams had significantly higher short‐axis systolic circumferential strain rate (~3 fold) and displacement (~2 fold) than WT offspring of WT dams. Adult WT offspring of transgenic dams presented with a significantly lower ejection fraction (6% and 8%) and fractional shortening (18% and 22%) than sham WT and nano‐TiO2 transgenic offspring, respectively. This may suggest that in offspring lacking the transgene, enhanced maternal antioxidant defense alone may not provide sufficient protection into adulthood. This study highlights the beneficial role of enhanced antioxidant defense (during fetal development and adulthood) on cardiac dysfunction following gestational ENM exposure.

Support or Funding Information

Supported by: R01 HL‐128485 (JMH), CFO for the Ohio Valley Whipkey Trust (JMH), R01‐ES015022 (TRN), AHA‐17PRE33660333 (QAH).
Changes in cerebral expression of ERK 1/2 related to commercial sweetener consumption in BALB/c mice
Authors: Contreras-Chavez Gerson Gabriel , Estrada José A. , Contreras Irazú
Published: April 2020

Non‐nutritive sweeteners are food additives that provide sweet taste with low energy content. Consumption of these compounds has become a common practice in the general population to reduce calorie intake from the diet. Nonetheless, there is evidence suggesting consumption of non‐nutritive sweeteners can induce changes in feeding behavior, altering energy intake, body weight and adiposity.

Previous studies have revealed that intake of sucralose and steviol glycosides lead to changes in the JAK/STAT3 signaling pathway in the brain of mice, altering food intake patterns, body weight and adiposity. However, the effect of these compounds on other signaling pathways, such as MAPK and ERK 1/2 in particular, which are also involved in the regulation of food intake in the central nervous system, has not been studied.

The main objective of this project is to determine the effects of frequent intake of sucrose, sucralose or steviol glycosides on the expression of total and phosphorylated ERK 1/2 protein in the brain of BALB/c mice.

To this end, 8 week‐old BALB/c male and female mice were supplemented with sucrose, sucralose or steviol glycosides for 6 weeks in their daily drinking water. Subsequently, total brain protein extracts were used to analyze the expression of total and phosphorylated ERK 1/2 proteins by western blot.

Our preliminary results show that expression of pERK was reduced in male mice supplemented steviol glycosides compared to non‐supplemented controls, while sucrose and sucralose groups has increased expression of pERK. In contrast, female animals supplemented with sucrose had decreased expression of pERK, which was increased in the steviol glycosides group compared to non‐supplemented controls. No differences were observed in total ERK expression among groups.

In conclusion, our preliminary results suggest that chronic consumption of non‐nutritive sweeteners may induce sex‐specific alterations in the ERK signaling pathway, with possible effects on feeding behavior and body weight in vivo.
The renal AE4 transporter (Slc4a9) is essential for the upregulation of pendrin (Slc26a4) and prevents life threatening hypochloremic metabolic alkalosis during base loading
Authors: Ehmke Heimo , Koch Mirijam , Huebner Christian A. , Meyer-Schwesinger Catherine , Vitzthum Helga
Published: April 2020

The renal transporter AE4 (Slc4a9) is localized to the basolateral membrane of type B intercalated cells (β‐ICs) in the collecting duct. Recently, it has been shown that β‐ICs not only contribute to renal HCO3− secretion, but also to renal sodium (Na+) reabsorption during dietary salt restriction. Currently it is generally assumed that AE4 constitutes the primary basolateral Na+ extrusion pathway in β‐ICs, but there is still no in vivo evidence of such a role. To test this hypothesis, we subjected AE4 knockout mice and their wild‐type littermates (WT) to either a salt (NaCl) deficient diet alone (10 days) or a salt deficient diet combined with an additional base load (230 mM NaHCO3, 7 days), and analyzed the effects of each diet on plasma volume, renal Na+ and Cl− handling, renal protein expression levels of NHE3, NCC, ENaC and pendrin, plasma renin and aldosterone levels, and acid‐base homeostasis. Surprisingly, AE4 knockout mice did not show any signs of Na+ loss or volume contraction or compensatory activation of other renal Na+ reabsorption pathways under salt deficient diet. However, we observed that AE4 knockout mice developed a mild hypochloremic metabolic alkalosis under these conditions, which severely aggravated when the animals were additionally subjected to a base load (NaHCO3). In contrast, WT littermates maintained normal Na+ and acid‐base balance under both diet regimes. While WT mice increased pendrin expression within the first day of base loading, this response was absent in AE knockout animals. These findings indicate that AE4 is dispensable for Na+ homeostasis during salt restriction, but is essential for the stimulatory effect of alkalosis on the expression of the apical Cl−/HCO3− exchanger pendrin. An insufficient function of AE4 facilitates the development of a life‐threatening hypochloremic metabolic alkalosis.
Purification and Characterization of a Putative Adenylosuccinate Lyase (Toy F) Involved In Toyocamycin Production
Authors: Subedi Subeksha , Bruender Nathan A.
Published: April 2020

The observed increase in bacteria resistant to a variety of antibiotics is starting to grow at an alarming rate and it poses a threat to humankind. This has led to a renewed interest in a variety of fields to discover and develop molecules with novel therapeutic mechanisms in order to combat these newly emerging strains of bacteria. Two molecules that show promise as novel antibiotics are Toyocamycin and Sangivamycin, which are produced by the bacteria Streptomyces rimosus. However, they are difficult molecules to synthesize in large quantities using standard synthetic approaches. An alternative approach to production of these molecules is using the enzymes from the organism. This chemoenzymatic approach has the main challenge of identifying the enzymes involved in the natural biosynthetic pathway of the metabolites. Early work by McCarty and Bandarian has identified a cluster of genes that encode putative enzymes proposed to carry out this synthesis in S. rimosus. The work presented in this poster focuses on the purification and functional characterization of the enzyme ToyF, which is a putative adenylosuccinate lyase. ToyF was shown to convert the substrate analog adenylosuccinate into AMP, which is consistent with its predicted function.
Prenatal chronic stimulation of the endocannabinoid system affects the respiratory motor outputs of juvenile male and female rats
Authors: Patrone Luis Gustavo A. , Karlen-Amaranre Marlusa , Bicego Kenia C. , Zoccal Daniel B. , Gargaglioni Luciane H.
Published: April 2020

The use of drugs of abuse during pregnancy can severely affect the development of physiological systems including the central respiratory network, mainly because prenatal period is extremely susceptible to external and pharmacological interventions that may cause postnatal consequences to the offspring. The endocannabinoid system is already present in the central nervous system in early stages of embryonic development, including in areas responsible for respiratory control. Nevertheless, the effect of external cannabinoids on the central cardiorespiratory network development as well as in the chemosensitivity and the future consequences during juvenile period is still unclear. Here, we evaluate the effects of prenatal exposure to cannabinoid on the respiratory motor outputs of juvenile (P27–28) male and female rats by using a decerebrated, arterially perfused in situ preparation. To this end, osmotic pumps were implanted subcutaneously in pregnant rats at embryonic day 0 and delivered vehicle (VEH) or CB1 receptor agonist (WIN 55212‐2, 0.5 mg/Kg/day) for 21 days. In situ preparations of juvenile rats were used for recording the respiratory motor and sympathetic outputs during normocapnia (5% CO2), hypercapnia (8% CO2) and stimulation of peripheral chemoreceptors with potassium cyanide (KCN; 25 μg in 50 μL). Our data showed that prenatal WIN exposure resulted in a tonic increase in phrenic burst amplitude in male animals, which was sustained during the KCN and CO2 challenge, while WIN‐treated females exhibited elevated phrenic burst frequency. The activity of the vagus nerve was also higher in male WIN‐treated animals, either under basal condition, KCN or hypercapnic exposure, although no effects were observed in the female group. The frequency of late‐expiratory events in abdominal activity, which indicate active expiration, was significantly higher (37%) for male treated animals during KCN stimulus, with no significant effects in the female group. In both sexes, prenatal WIN treatment did not modify the levels of thoracic sympathetic activity. These results suggest that prenatal chronic stimulation of endocannabinoid system promotes alterations in the respiratory network development affecting the activity of nerves responsible for the ventilatory motor drive during juvenile period. These effects seem to be potentiated in males.

Support or Funding Information

FAPESP and CNPq
Renal Protection against Post Ischemia‐reperfusion Injury in Fatty Acid Amide Hydrolase Knockout Mice
Authors: Chen Chaoling , Wang Weili , Hu Gaizun , Xie Dengpiao , Ritter Joseph , Li Ningjun
Published: April 2020

Renal fibrosis is a typical characteristic of progressive chronic kidney disease (CKD). Current treatment options to slow renal fibrosis are still very limited. Endocannabinoids known as anandamide and 2‐arachidonoylglycerol play a critical role in regulating renal physiology via cannabinoid receptors (CB1 and CB2). Fatty acid amide hydrolase (FAAH) is one of the major enzymes to metabolize endocannabinoids. Studies have shown that endocannabinoid system is present in the kidney and that CB1 and CB2 are involved in CKD. However, the role of FAAH in CKD remains unclear. The present study tested the hypothesis that FAAH pathway participates in the kidney damage in post ischemia‐reperfusion (PIR) model, which produces chronic renal injury and fibrosis, using FAAH knockout mice. Male wild type C57BL6 (WT) and Faah−/− mice were subjected to PIR by unilateral ischemia‐reperfusion and contralateral nephrectomy 10 days after ischemia‐reperfusion. Compared with WT mice, Faah−/− mice showed significantly reduced BUN (mean: 22.0 ± 1.16, 102.0± 9.50, and 61± 4.35 mg/dL in WT‐shame, WT‐PIR, and Faah−/−‐PIR mice, respectively, p<0.05) and plasma creatinine level (mean: 0.18 ± 0.02, 1.18 ± 0.06, and, 0.54± 0.05 mg/dL in WT‐shame, WT‐PIR, and Faah−/−‐PIR mice, respectively, p<0.05) in response to PIR. Western blot analysis showed that increased expression of fibrotic protein alpha‐smooth muscle actin in the kidneys were significantly reduced in Faah−/− mice, whereas remained high in WT mice under PIR (1.85‐fold versus 2.46‐fold, p<0.05). Collagen staining by Sirius Red showed the same pattern as alpha‐smooth muscle actin expression in the kidneys from different groups of mice. Histological evaluation by Periodic acid–Schiff (PAS) staining showed that Faah−/− mice exhibited less tissue damage especially in tubular structure in response to PIR compared with WT mice. These results suggest that inhibition of FAAH may preserve renal function and prevent renal fibrosis and that FAAH may serve as a target for developing novel and effective treatment for CKD.

Support or Funding Information

NIH grants R01DK54927, R01DK107991, R01HL145163
Comparative Anticoagulant Effects of Parenteral Sulodexide as Measured by Activated Clotting Time. Relevance to Dosing in Various Indications
Authors: Daravath Bharathi , Fareed Jawed , Iqbal Omer , Kouta Ahmed , Hoppensteadt Debra , Siddiqui Fakiha , Lewis Mary , Lewis Bruce
Published: April 2020

Introduction

Sulodexide is a mucosal tissue derived glycosaminoglycan of mammalian origin which is clinically used in oral (capsule) and parenteral (ampules) forms for various clinical indications. The biologic potency of sulodexide is expressed in lipoproteinlipase releasing units (LSU). As a parenteral drug sulodexide produces sizable anticoagulant effects in various laboratory assays including the activated clotting time which is routinely used during surgical procedures. In comparison to heparin the USP referenced potency of sulodexide is reported to be 95 – 105 U/mg by anti‐Xa assay and 30 – 40 U/mg by anti‐IIa assay. The purpose of this study is to determine the whole blood anticoagulant effect of parenteral preparations of sulodexide and its neutralization by protamine sulfate.

Materials and Methods

Parenteral formulations (ampules) containing 600 LSU/2 ml were diluted with saline to make 10 and 100 LSU/ml working solutions. Protamine sulfate was obtained from the Loyola hospital pharmacy and diluted to 1.00 – 0.12 mg/ml. Anticoagulant effects of sulodexide over the concentration range of 1.0 – 0.25 LSU/ml were measured on the Hemachrom ACT instrument in freshly drawn normal human blood (n=5–10). Protamine neutralization studies were carried out at a fixed sulodexide level (1.0 LSU/ml) and variable protamine sulfate levels (50–12.5 μg/ml).

Results

Sulodexide produced a concentration‐dependent anticoagulant effect in normal human whole blood over a concentration range of 0.25 – 1.0 LSU/ml in the range of (238 ± 24 to 401 ± 30 seconds) in comparison to saline control (136 ± 12 seconds). Protamine sulfate supplementation at a concentration range of 50 – 12.5 μg/ml produced varying levels of neutralization of the anticoagulant effects of sulodexide (1 LSU/ml). 50 μg/ml protamine completely neutralized sulodexide at 1 LSU/ml whereas at lower concentrations it partially neutralized the anticoagulant effects.

Discussion

The results demonstrate that sulodexide in parenteral formulation produces a strong anticoagulant effect as measured by whole blood ACT. At a concentration of 1.0 LSU/ml, sulodexide produces an anticoagulant effect that is comparable to that observed with surgical anticoagulation levels of heparin (>3 μg/ml). The anticoagulant effects of sulodexide are neutralized by protamine sulfate which can be used as an antidote for sulodexide.

Conclusion

Parenteral (ampules) sulodexide can be used for anticoagulation in dose dependent fashion and protamine sulfate can be used as an antidote for this agent thus in the event of a shortage of unfractionated heparin sulodexide can be used as a substitute anticoagulant drug for the indications in which heparin is considered.
Using Exogenous βHB as an Epigenetic Modifier to Mitigate the Symptoms of Kabuki Syndrome
Authors: Moss Sara , Soliven Maricel , Poff Angela , D’Agostino Dominic
Published: April 2020

Background

Kabuki Syndrome is a rare genetic disorder that leads to many developmental abnormalities, and is caused by a heterozygous mutation in either KMT2D (Type 1) or KDM6A (Type 2) that leads to a loss of function. Both genes play a role in gene regulation via histone modification. KMT2D is a gene that codes for the protein kmt2d, a lysine methyltransferase responsible for methylating H3K4. The KDM6A gene encodes the protein kdm6a, a demethylase responsible for demethylating H3K27. Both gene functions contribute to the opening of the chromatin. When either of these two proteins are deficient, craniofacial, skeletal, mental, and dermatologic development are severely impacted. Other developmental processes are impacted, but to varying degrees across patients. The endogenous ketone metabolite beta‐hydroxybutyrate (βHB) is a known epigenetic modifier, functioning as a class I and IIa histone deacetylase inhibitor. Previous research has shown that the ketogenic diet increases H3K4 acetylation and methylation rates, making βHB a potential therapeutic intervention for Kabuki syndrome. Additionally, H3K4 is a beta‐hydroxybutyrylation site, further underlying the potential for βHB to affect the epigenetic modifications observed in the disease. However, there is no current research on beta‐hydroxybutyrylation in Kabuki, nor on the effects of continuous exogenous βHB supplementation in the disorder.

Methods

Kmt2d+/βGeo (Type 1 Kabuki syndrome model) mice were fed standard diet with or without 15% BHB‐MCT supplementation for 12–15 weeks. Blood ketones, glucose, and body weight were monitored during the treatment period. Open field, Elevated Plus Maze, Novel Object Recognition, and Grip Strength behavioral tests were performed at the end of treatment. Various tissues were harvested, including the brain, for ongoing ex vivo analysis of markers associated with learning, memory, and development.

Conclusions

The 15% BHB‐MCT supplementation is sufficient to raise blood ketone levels in Kmt2d+/βGeo mice. Behavioral and molecular analysis is ongoing.

Support or Funding Information

Disruptive Nutrition, Florida high Tech Corridor